Anti-ANGPTL3 Antibodies and Uses Thereof

ABSTRACT

A fully human antibody or antigen-binding fragment of a human antibody that specifically binds and inhibits or interferes with at least one activity of human angiopoietin-like protein 3 (hANGPTL3) is provided. The human anti-hANGPTL3 antibodies are useful in treating diseases or disorders associated with ANGPTL3, such as hyperlipidemia, hyperlipoproteinemia and dyslipidemia, including hypertriglyceridemia, hypercholesterolemia, chylomicronemia, and so forth. Furthermore, the anti-hANGPTL3 antibodies can be administered to a subject in need thereof to prevent or treat diseases or disorders, for which abnormal lipid metabolism is a risk factor. Such diseases or disorders include cardiovascular diseases, such as atherosclerosis and coronary artery diseases; acute pancreatitis; nonalcoholic steatohepatitis (NASH); diabetes; obesity; and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application Nos. 61/498,518, filed on Jun. 17, 2011; and61/578,309, filed on Dec. 21, 2011, the disclosures of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is related to human antibodies and antigen-bindingfragments of human antibodies that specifically bind humanangiopoietin-like protein 3 (hANGPTL3), and therapeutic methods of usingthose antibodies.

BACKGROUND

The angiopoietin-like protein 3 (ANGPTL3) gene was identified from theEST database based on signal sequences and amphipathic helices, and afull-length ANGPTL3 cDNA was subsequently isolated from a human fetalliver/spleen cDNA library (Conklin et al., 1999, Genomics 62: 477-482).The deduced 460-amino acid hANGPTL3 protein shares 76% amino acidsequence identity with mouse ANGPTL3 and has the characteristicstructure of angiopoietins; i.e., a signal peptide, an extended helicaldomain predicted to form dimeric or trimeric coiled-coils, a shortlinker peptide, and a globular fibrinogen homology domain (FD) (Conklinet al., 1999, supra). ANGPTL3 contains the 4 conserved cysteine residuesimplicated in the intramolecular disulfide bonds within the FD; however,ANGPTL3 contains neither the two additional cysteines nor thecharacteristic calcium-binding motif found in the FDs of angiopoietins(ANGs; i.e., ANG1, ANG2 and ANG4) (Conklin et al., 1999, supra), whichare protein growth factors that promote angiogenesis. In addition,unlike ANGs, ANGPTL3 does not bind to Tie2; however, it may also induceangiogenesis by binding to integrin α_(v)β₃ via its C-terminal FD(Camenisch et al., 2002, J Biol Chem 277:17281-17290).

Comprehensive in vivo data were obtained from the outbred KK mousemodel, which is moderately obese with abnormally high levels of plasmainsulin, glucose, and lipids, resembling type 2 diabetes mellitus inhumans (Koishi et al., 2002, Nature Genetics 30:151-157). One sub-strainof mouse, the KK/San, however, was found to exhibit abnormally lowplasma lipid levels (hypolipidemia), which were inherited as a Mendelianrecessive. The loci was mapped to chromosome 4 and eventually identifiedto be the gene encoding ANGPTL3, which contained a 4-bp nucleotidesequence insertion in exon 6 (Koishi et al., 2002, supra). Conversely,plasma lipid levels increase after adenovirus-mediated transfer ofANGPTL3 gene, or after administration of recombinant human ANGPTL3 inKK/San mice. This effect was not mediated by changes in genes involvedin cholesterol synthesis, lipoprotein clearance or NEFA oxidation(Koishi et al., 2002, supra). Further, in vitro analysis of recombinantprotein showed that ANGPTL3 directly inhibits lipoprotein lipase (LPL)activity, indicating that it is a lipid metabolism modulator thatregulates very low density lipoprotein (VLDL) triglyceride levelsthrough the inhibition of LPL activity (Shimizugawa et al., 2002, J BiolChem 277(37):33742-33748). It has been shown that the N-terminalcoiled-coil domain, especially the N-terminal region residues 17-165,and not the C-terminal FD, of ANGPTL3, is required for its activity ofincreasing plasma triglyceride levels in mice (Ono et al., 2003, J BiolChem 278:41804-41809).

The amino acid and nucleotide sequences of human ANGPTL3 are shown inSEQ ID NOS:161 and 162, respectively. Antibodies to ANGPTL3 aredisclosed in, for example, WO2008/073300 and U.S. Pat. No. 7,935,796.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides fully human monoclonalantibodies (mAbs) and antigen-binding fragments thereof thatspecifically bind and neutralize, inhibit, block, abrogate, reduce orinterfere with, at least one activity of ANGTPL3, in particular, humanANGPTL3 (SEQ ID NO:161). The activity of ANGPTL3 that can beneutralized, inhibited, blocked, abrogated, reduced or interfered with,by the antibodies or fragments thereof of the invention, includes, butnot by the way of limitation, inhibition of LPL activity, induction ofangiogenesis, and the like. In one embodiment, an antibody or fragmentthereof of the present invention can neutralize, inhibit, block,abrogate, reduce or interfere with, an activity of hANGPTL3 by bindingto an epitope of hANGPTL3 that is directly involved in the targetedactivity of hANGPTL3. In another embodiment, an antibody or fragmentthereof of the invention can neutralize, inhibit, block, abrogate,reduce or interfere with, an activity of hANGPTL3 by binding to anepitope of hANGPTL3 that is not directly involved in the targetedactivity of hANGPTL3, but the antibody or fragment binding theretosterically or conformationally inhibits, blocks, abrogates, reduces orinterferes with, the targeted activity of hANGPTL3. In yet anotherembodiment, an antibody or fragment thereof of the invention binds to anepitope of hANGPTL3 that is not directly involved in the targetedactivity (e.g., inhibiting LPL activity, inducing angiogenesis, and thelike) of hANGPTL3 (i.e., a non-blocking antibody), but the antibody orfragment binding thereto results in the enhancement of the clearance ofhANGPTL3 from the circulation, compared to the clearance of hANGPTL3 inthe absence of the antibody or fragment thereof, thereby indirectlyinhibiting, blocking, abrogating, reducing or interfering with, anactivity of hANGPTL3. Clearance of hANGPTL3 from the circulation can beparticularly enhanced by combining two or more different non-blockingantibodies that do not compete with one another for specific binding tohANGPTL3.

The antibodies (Abs) can be full-length (for example, an IgG1 or IgG4antibody) or may comprise only an antigen-binding portion (for example,a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody comprising a heavy chainvariable region (HCVR) selected from the group consisting of SEQ IDNO:2, 18, 34, 50, 66, 82, 98, 114, 130, 146 and 180, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity. In another embodiment, the antibodyor antigen-binding fragment thereof comprises a HCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO:2, 18, 34,66, 82, 114, and 180. In yet another embodiment, the antibody or anantigen-binding fragment thereof comprises a HCVR having an amino acidsequence of SEQ ID NO:66.

In one embodiment, an antibody or antigen-binding fragment of anantibody comprises a light chain variable region (LCVR) selected fromthe group consisting of SEQ ID NO:10, 26, 42, 58, 74, 90, 106, 122, 138,154 and 188, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity. Inanother embodiment, the antibody or antigen-binding portion of anantibody comprises a LCVR having an amino acid sequence selected fromthe group consisting of SEQ ID NO:10, 26, 42, 74, 90, 122 and 188. Inyet another embodiment, the antibody or antigen-binding portion of anantibody comprises a LCVR having an amino acid sequence of SEQ ID NO:74.

In further embodiments, the antibody or fragment thereof comprises aHCVR and LCVR sequence pair (HCVR/LCVR) selected from the groupconsisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106,114/122, 130/138, 146/154 and 180/188. In one embodiment, the antibodyor fragment thereof comprises a HCVR and LCVR sequence pair selectedfrom the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 66/74, 82/90,114/122 and 180/188. In another embodiment, the antibody or fragmentthereof comprises a HCVR and LCVR sequence pair of SEQ ID NO:66/74.

In a second aspect, the invention features an antibody orantigen-binding fragment of an antibody comprising a heavy chaincomplementarity determining region 3 (HCDR3) amino acid sequenceselected from the group consisting of SEQ ID NO:8, 24, 40, 56, 72, 88,104, 120, 136, 152 and 186, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a light chain CDR3 (LCDR3) amino acid sequence selectedfrom the group consisting of SEQ ID NO:16, 32, 48, 64, 80, 96, 112, 128,144, 160 and 194, or substantially similar sequences thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.In one embodiment, the antibody or fragment thereof comprises aHCDR3/LCDR3 amino acid sequence pair comprising SEQ ID NO:8/16, 24/32,40/48, 56/64, 72/80, 88/96, 104/112, 120/128, 136/144, 152/160 or186/194. In another embodiment, the antibody or fragment thereofcomprises a HCDR3/LCDR3 amino acid sequence pair comprising SEQ IDNO:8/16, 24/32, 40/48, 72/80, 88/96, 120/128 or 186/194. In yet anotherembodiment, the antibody or fragment thereof comprises a HCDR3/LCDR3amino acid sequence pair comprising SEQ ID NO:72/80.

In a further embodiment, the antibody or fragment thereof furthercomprises a heavy chain CDR1 (HCDR1) amino acid sequence selected fromthe group consisting of SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132,148 and 182, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; and aheavy chain CDR2 (HCDR2) amino acid sequence selected from the groupconsisting of SEQ ID NO:6, 22, 38, 54, 70, 86, 102, 118, 134, 150 and184, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; andoptionally further comprises a light chain CDR1 (LCDR1) amino acidsequence selected from the group consisting of SEQ ID NO:12, 28, 44, 60,76, 92, 108, 124, 140, 156 and 190, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; and/or a light chain CDR2 (LCDR2) amino acid sequenceselected from the group consisting of SEQ ID NO:14, 30, 46, 62, 78, 94,110, 126, 142, 158 and 192, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

Alternatively, the invention features an antibody or antigen-bindingfragment of an antibody comprising a HCDR1/HCDR2/HCDR3 combinationselected from the group consisting of SEQ ID NO:4/6/8, 20/22/24,36/38/40, 52/54/56, 68/70/72, 84/86/88, 100/102/104, 116/118/120,132/134/136, 148/150/152 and 182/184/186; and/or a LCDR1/LCDR2/LCDR3combination selected from the group consisting of SEQ ID NO:12/14/16,28/30/32, 44/46/48, 60/62/64, 76/78/80, 92/94/96, 108/110/112,124/126/128, 140/142/144, 156/158/160 and 190/192/194. In oneembodiment, the heavy and light chain CDR amino acid sequences comprisea CDR sequence combination selected from the group consisting of SEQ IDNO:4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48,52/54/56/60/62/64, 68/70/72/76/78/80, 84/86/88/92/94/96,100/102/104/108/110/112, 116/118/120/124/126/128,132/134/136/140/142/144, 148/150/152/156/158/160 and182/184/186/190/192/194. In one embodiment, the heavy and light chainCDR amino acid sequences comprise a CDR sequence combination of SEQ IDNO: 4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48,68/70/72/76/78/80, 84/86/88/92/94/96, 116/118/120/124/126/128 or182/184/186/190/192/194. In another embodiment, the heavy and lightchain CDR amino acid sequences comprise a CDR sequence combination ofSEQ ID NO:68/70/72/76/78/80.

In a related embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody which specifically bindshANGPTL3, wherein the antibody or fragment thereof comprises heavy andlight chain CDR domains contained within HCVR/LCVR pairs selected fromthe group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74,82/90, 98/106, 114/122, 130/138, 146/154 and 180/188. Methods andtechniques for identifying CDRs within HCVR and LCVR amino acidsequences are known in the art and can be applied to identify CDRswithin the specified HCVR and/or LCVR amino acid sequences disclosedherein. Conventional definitions that can be applied to identify theboundaries of CDRs include the Kabat definition, the Chothia definition,and the AbM definition. In general terms, the Kabat definition is basedon sequence variability, the Chothia definition is based on the locationof the structural loop regions, and the AbM definition is a compromisebetween the Kabat and Chothia approaches. See, e.g., Kabat, “Sequencesof Proteins of Immunological Interest,” National Institutes of Health,Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948(1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272(1989). Public databases are also available for identifying CDRsequences within an antibody. In one embodiment, the antibody orfragment thereof comprises CDR sequences contained within a HCVR andLCVR pair of SEQ ID NO: 2/10, 18/26, 34/42, 66/74, 82/90, 114/122 or180/188. In another embodiment, the antibody or fragment thereofcomprises CDR sequences contained within a HCVR and LCVR pair of SEQ IDNO:66/74.

In another related embodiment, the invention provides an antibody orantigen-binding fragment thereof that competes for specific binding tohANGPTL3 with an antibody or antigen-binding fragment comprising heavyand light chain CDR sequences contained in a HCVR/LCVR sequence pair ofSEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154 or 180/188. In one embodiment, the antibody orantigen-binding fragment of the invention competes for specific bindingto hANGPTL3 with an antibody or fragment thereof comprising a HCVR/LCVRsequence pair of SEQ ID NO:66/74. In another embodiment, the antibody orantigen-binding fragment of the invention competes for specific bindingto hANGPTL3 with an antibody or fragment thereof comprising a heavy andlight chain CDR sequence combination selected from the group consistingof 4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48,52/54/56/60/62/64, 68/70/72/76/78/80, 84/86/88/92/94/96,100/102/104/108/110/112, 116/118/120/124/126/128,132/134/136/140/142/144, 148/150/152/156/158/160 and182/184/186/190/192/194. In one embodiment, the antibody orantigen-binding fragment thereof of the invention competes for specificbinding to hANGPTL3 with an antibody or fragment thereof comprising aheavy and light chain CDR sequence combination of SEQ IDNOS:68/70/72/76/78/80.

In another related embodiment, the invention provides an antibody orantigen-binding fragment thereof that binds the same epitope on hANGPTL3that is recognized by an antibody or fragment thereof comprising heavyand light chain CDR sequences from a HCVR/LCVR sequence pair of SEQ IDNO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138,146/154 or 180/188. In one embodiment, the antibody or antigen-bidingfragment of the invention binds the same epitope on hANGPTL3 as thatrecognized by the antibody or fragment thereof comprising a HCVR/LCVRsequence pair of SEQ ID NO:66/74. In one embodiment, the antibody orfragment thereof of the invention binds the same epitope on hANGPTL3that is recognized by an antibody or fragment thereof comprising a heavyand light chain CDR sequence combination selected from the groupconsisting of 4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48,52/54/56/60/62/64, 68/70/72/76/78/80, 84/86/88/92/94/96,100/102/104/108/110/112, 116/118/120/124/126/128,132/134/136/140/142/144, 148/150/152/156/158/160 and182/184/186/190/192/194. In one embodiment, such an epitope isrecognized by an antibody or fragment thereof comprising a heavy andlight chain CDR sequence combination of SEQ ID NO:68/70/72/76/78/80.

In a third aspect, the invention features an isolated anti-hANGPTL3antibody or antigen-binding fragment thereof that binds to an epitopesituated within the N-terminal coiled-coil region at residues 17 to 209of SEQ ID NO:161 and neutralizes, inhibits, abrogates, reduces orinterferes with, at least one activity of hANGPTL3. In anotherembodiment, the invention provides an isolated antibody orantigen-binding fragment of an antibody that specifically binds to anepitope situated within the N-terminal coiled-coil region of hANGPTL3(SEQ ID NO:161) and neutralizes, inhibits, abrogates, reduces orinterferes with, at least one activity of hANGPTL3, with the provisothat the antibody or fragment thereof does not bind to the ANGPTL3peptide of SEQ ID NO:170 (corresponds to residues Glu32 to Leu57 ofhANGPTL3 of SEQ ID NO:161). In one embodiment, the antibody or fragmentthereof of the invention specifically binds to an epitope withinresidues 17 to 200, 17 to 100, 17 to 70, 17 to 65, 17 to 60, 17 to 57,or 17 to 50, of hANGPTL3 (SEQ ID NO:161), optionally with the provisothat the antibody or fragment thereof does not bind to the ANGPTL3peptide of SEQ ID NO:170. In another embodiment, the antibody orfragment thereof specifically binds to an epitope within residues 40 to200, 40 to 100, 40 to 70, 50 to 200, 50 to 100, 50 to 70, 58 to 200, 58to 100, 58 to 70, 58 to 68, or 61 to 66, of hANGPTL3 (SEQ ID NO:161),optionally with the proviso that the antibody or fragment thereof doesnot bind to the ANGPTL3 peptide of SEQ ID NO:170. In some embodiments,the antibody or antibody fragment binds an epitope which may involvemore than one of the enumerated epitopes or residues within theN-terminal coiled-coil region of hANGPTL3, optionally with the provisothat the antibody or fragment thereof does not bind to the ANGPTL3peptide of SEQ ID NO:170.

In a fourth aspect, the invention provides nucleic acid moleculesencoding anti-ANGPTL3 antibodies or fragments thereof, in particular,any one of those described above. Recombinant expression vectorscarrying the nucleic acids of the invention, and host cells, e.g.,bacterial cells, such as E. coli, or mammalian cells, such as CHO cells,into which such vectors have been introduced, are also encompassed bythe invention, as are methods of producing the antibodies by culturingthe host cells under conditions permitting production of the antibodies,and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113,129, 145 and 179, or a substantially identical sequence having at least90%, at least 95%, at least 98%, or at least 99% homology thereof. Inanother embodiment, the antibody or fragment thereof comprises a HCVRencoded by a nucleic acid sequence of SEQ ID NO:1, 17, 33, 65, 81, 113or 179. In yet another embodiment, the antibody or fragment thereofcomprises a HCVR encoded by a nucleic acid sequence of SEQ ID NO:65.

In one embodiment, an antibody or antigen-binding fragment thereofcomprises a LCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153and 187, or a substantially identical sequence having at least 90%, atleast 95%, at least 98%, or at least 99% homology thereof. In anotherembodiment, the antibody or fragment thereof comprises a LCVR encoded bya nucleic acid sequence of SEQ ID NO:9, 25, 41, 73, 89, 121 or 187. Inyet another embodiment, the antibody or fragment thereof comprises aLCVR encoded by a nucleic acid sequence of SEQ ID NO:73.

In further embodiments, the antibody or fragment thereof comprises aHCVR and LCVR (HCVR/LCVR) sequence pair encoded by a nucleic acidsequence pair selected from the group consisting of SEQ ID NO:1/9,17/25, 33/41, 49/57, 65/73, 81/89, 97/105, 113/121, 129/137, 145/153 and179/187. In one embodiment, the antibody or fragment thereof comprises aHCVR/LCVR sequence pair encoded by a nucleic acid sequence pair of SEQID NO:1/9, 17/25, 33/41, 65/73, 81/89, 113/121 or 179/187. In anotherembodiment, the antibody or fragment thereof comprises a HCVR/LCVRsequence pair encoded by a nucleic acid sequence pair of SEQ IDNO:65/73.

In one embodiment, the invention features an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:7,23, 39, 55, 71, 87, 103, 119, 135, 151 and 185, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% homology thereof; and a LCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:15,31, 47, 63, 79, 95, 111, 127, 143, 159 and 193, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% homology thereof. In one embodiment, the antibody orfragment thereof comprises a HCDR3 and LCDR3 sequence pair encoded bythe nucleic acid sequence pair selected from the group consisting of SEQID NO:7/15, 23/31, 39/47, 55/63, 71/79, 87/95, 103/111, 119/127,135/143, 151/159 and 185/193. In another embodiment, the antibody orfragment thereof comprises a HCDR3 and LCDR3 sequence pair encoded bythe nucleic acid sequence pair of SEQ ID NO:7/15, 23/31, 39/47, 71/79,87/95, 119/127 or 185/193. In yet another embodiment, the antibody orfragment thereof comprises a HCDR3 and LCDR3 sequence pair encoded bythe nucleic acid sequence pair of SEQ ID NO:71/79.

In a further embodiment, the antibody or fragment thereof furthercomprises a HCDR1 domain encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131,147 and 181, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% homology thereof; and aHCDR2 domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO:5, 21, 37, 53, 69, 85, 101, 117, 133, 149 and183, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; and optionallyfurther comprises a LCDR1 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO:11, 27, 43, 59, 75, 91,107, 123, 139, 155 and 189, or a substantially identical sequence havingat least 90%, at least 95%, at least 98%, or at least 99% homologythereof; and/or a LCDR2 domain encoded by a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:13, 29, 45, 61, 77, 93, 109, 125,141, 157 and 191, or a substantially identical sequence having at least90%, at least 95%, at least 98%, or at least 99% homology thereof.

Alternatively, the invention features an antibody or antigen-bindingfragment of an antibody comprising a HCDR1/HCDR2/HCDR3 combinationencoded by a nucleotide sequence combination selected from the groupconsisting of SEQ ID NO:3/5/7, 19/21/23, 35/37/39, 51/53/55, 67/69/71,83/85/87, 99/101/103, 115/117/119, 131/133/135, 147/149/151 and181/183/185; and/or a LCDR1/LCDR2/LCDR3 combination encoded by anucleotide sequence combination selected from the group consisting ofSEQ ID NO:11/13/15, 27/29/31, 43/45/47, 59/61/63, 75/77/79, 91/93/95,107/109/111, 123/125/127, 139/141/143, 155/157/159 and 189/191/193. Inone embodiment, the antibody or fragment thereof comprises heavy andlight chain CDR sequences encoded by a nucleotide sequence combinationof SEQ ID NO:67/69/71/75/77/79.

In a fifth aspect, the invention features a human anti-ANGPTL3 antibodyor antigen-binding fragment thereof comprising a heavy chain variableregion (HCVR) encoded by nucleotide sequence segments derived fromV_(H), D_(H) and J_(H) germline sequences, and a light chain variableregion (LCVR) encoded by nucleotide sequence segments derived from V_(K)and J_(K) germline sequences, wherein the HCVR and the LCVR are encodedby nucleotide sequence segments derived from a germline gene combinationselected from the group consisting of: (i) V_(H)3-43, D_(H)3-3, J_(H)3,V_(K)1-5 and J_(K)2; (ii) V_(H)3-11, D_(H)1-1, J_(H)4, V_(K)1-39 andJ_(K)4; (iii) V_(H)3-30, D_(H)1-7, J_(H)6, V_(K)1-5 and J_(K)1; (iv)V_(H)3-30, D_(H)1-26, J_(H)6, V_(K)1-12 and J_(K)3; (v) V_(H)3-30,D_(H)3-10, J_(H)6, V_(K)1-12 and J_(K)3; and (vi) V_(H)3-23, D_(H)3-10,J_(H)4, V_(K)1-5 and J_(K)1.

In a sixth aspect, the invention features an antibody or antigen-bindingfragment thereof that specifically binds to hANGPTL3 with an equilibriumdissociation constant (K_(D)) of about 7 nM or less, about 6 nM or less,about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nMor less, or about 1 nM or less, as measured by surface plasmon resonanceassay (for example, BIACORE™). In certain embodiments, the antibody ofthe invention exhibits a K_(D) of about 800 μM or less, about 700 μM orless; about 600 μM or less; about 500 μM or less; about 400 μM or less;about 300 μM or less; about 200 μM or less; about 100 μM or less; orabout 50 μM or less.

In a seventh aspect, the present invention provides an anti-hANGPTL3antibody or antigen-binding fragment thereof that binds hANGPTL3 proteinof SEQ ID NO:161, but does not cross-react with a related protein, suchas a human angiopoietin-like protein 4 (hANGPTL4; SEQ ID NO:164), asdetermined by, for example, ELISA, surface plasmon resonance assay, orLUMINEX® XMAP® Technology, as described herein. ANGPTL4 is anothersecreted protein that is known to reduce LPL activity and has anN-terminal coiled-coil region and a C-terminal fibrinogen-like domain(Ge et al., 2004, J Biol Chem 279:2038-2045; Yau et al., 2009, J BiolChem 284:11942-11952). In related embodiments, the invention provides ananti-hANGPTL3 antibody or antigen binding fragment thereof that binds ahANGPTL3 protein and cross-reacts with a hANGPTL4 protein. In certainembodiments, the binding affinity of the hANGPTL3 antibody or fragmentthereof to hANGPTL4 protein is about 75% or less, or about 50% or less,of the binding affinity of the antibody or fragment to the hANGPTL3protein.

In another related embodiment, the invention provides an anti-hANGPTL3antibody or antigen binding fragment thereof that does not cross-reactwith mouse ANGPTL3 (mANGPTL3; SEQ ID NO:163), or rat ANGPTL3 (rANGPTL3;SEQ ID NO:175), but does cross-react with cynomolgus monkey (Macacafascicularis) ANGPTL3 (MfANGPTL3), for example, with the N-terminal17-170 residues of SEQ ID NO:177 (a partial amino acid sequence ofMfANGPTL3). In yet another related embodiment, the invention provides ananti-hANGPTL3 antibody or fragment thereof that cross-reacts withMfANGPTL3, mANGPTL3 and rANGTPL3.

The invention encompasses anti-hANGPTL3 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or e.g., removal of afucose moiety to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, removal of N-glycosylation site may reduce undesirableimmune reactions against the therapeutic antibodies, or increaseaffinities of the antibodies. In yet other applications, modification ofgalactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In an eighth aspect, the invention features a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds hANGPTL3 and a pharmaceutically acceptable carrier.In one embodiment, the invention provides a pharmaceutical compositioncomprising one or more anti-ANGPTL3 antibodies or fragments thereof ofthe invention, which do not cross-compete with one another, and apharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition of the invention can contain two or morenon-blocking antibodies, which do not compete with one another forspecific binding to hANGPTL3 and are effective in clearing hANGPTL3 fromthe circulation. Suitable combinations of non-blocking antibodiesinclude, but are not limited to, a combination of antibodies comprisingHCVR and LCVR sequence pairs (HCVR/LCVR) of: (i) SEQ ID NO:82/90 and180/188, respectively; (ii) SEQ ID NO:114/122 and 180/188, respectively;(iii) SEQ ID NO:82/90 and 18/26, respectively; or (iv) SEQ ID NO:114/122and 18/26, respectively.

In related embodiments, the invention features a composition which is acombination of an antibody or antigen-binding fragment thereof of theinvention, and a second therapeutic agent. The second therapeutic agentmay be one or more of any agent such as (1)3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors,such as cerivastatin, atorvastatin, simvastatin, pitavastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin, and the like; (2)inhibitors of cholesterol uptake and/or bile acid re-absorption; (3)niacin, which increases lipoprotein catabolism; (4) fibrates oramphipathic carboxylic acids, which reduce low-density lipoprotein (LDL)level, improve high-density lipoprotein (HDL) and TG levels, and reducethe number of non-fatal heart attacks; and (5) activators of the LXRtranscription factor that plays a role in cholesterol elimination suchas 22-hydroxycholesterol, or fixed combinations such as ezetimibe plussimvastatin; a statin with a bile resin (e.g., cholestyramine,colestipol, colesevelam), a fixed combination of niacin plus a statin(e.g., niacin with lovastatin); or with other lipid lowering agents suchas omega-3-fatty acid ethyl esters (for example, omacor). Furthermore,the second therapeutic agent can be one or more other inhibitors ofANGPTL3 as well as inhibitors of other molecules, such as ANGPTL4,ANGPTL5, ANGPTL6 and proprotein convertase subtilisin/kexin type 9(PCSK9), which are involved in lipid metabolism, in particular,cholesterol and/or triglyceride homeostasis. Inhibitors of thesemolecules include small molecules and antibodies that specifically bindto these molecules and block their activity.

In related embodiments, the second therapeutic agent may be one or moreanti-cancer agents, such as chemotherapeutic agents, anti-angiogenicagents, growth inhibitory agents, cytotoxic agents, apoptotic agents,and other agents well known in the art to treat cancer or otherproliferative diseases or disorders, as well as other therapeuticagents, such as analgesics, anti-inflammatory agents, includingnon-steroidal anti-inflammatory drugs (NSAIDS), such as Cox-2inhibitors, and the like, so as to ameliorate and/or reduce the symptomsaccompanying the underlying cancer/tumor.

In a ninth aspect, the invention features methods for neutralizing,inhibiting, blocking, abrogating, reducing or interfering with, hANGPTL3activity using one or more anti-hANGPTL3 antibodies or antigen-bindingfragments thereof of the invention. In one embodiment, the inventionprovides a therapeutic method comprising administering to a subject inneed thereof a therapeutically effective amount of a pharmaceuticalcomposition comprising one or more anti-hANGPTL3 antibodies orantigen-binding fragments thereof of the invention and, optionally oneor more additional therapeutic agents described above. The anti-ANGPTL3antibodies or fragments thereof of the invention may be neutralizingantibodies or non-blocking antibodies against ANGPTL3, or combinationsthereof.

In related embodiments, the invention provides methods of enhancing theclearance of hANGPTL3 from the circulation of a subject in need thereof,comprising administering to the subject at least two anti-hANGPTL3antibodies or fragments thereof of the invention that do not competewith one another for binding to hANGPTL3 and preferably do not block atleast one activity of hANGPTL3 (i.e., non-blocking antibodies). At leastone activity of hANGPTL3 referred to includes, but not the way oflimitation, inhibiting LPL activity, inducing angiogenesis, and thelike. In one embodiment, a combination of at least two non-blockinganti-hANGPTL3 antibodies or fragments thereof enhances the clearance ofhANGPTL3 from the circulation by at least about 20%, about 30%, about40%, about 50%, about 60%, about 70%, or about 80%, relative to notadministering the antibodies or fragments. Circulating levels ofhANGPTL3 can be measured by in vitro assays well known in the art andthose described herein. In another embodiment, the combination of atleast two non-blocking anti-hANGPTL3 antibodies comprises HCVR and LCVRsequence pairs (HCVR/LCVR) of: (i) SEQ ID NO:82/90 and 180/188,respectively; (ii) SEQ ID NO:114/122 and 180/188, respectively; (iii)SEQ ID NO:82/90 and 18/26, respectively; or (iv) SEQ ID NO:114/122 and18/26, respectively.

The disease or disorder treatable by the methods of the invention is anydisease or condition which is improved, ameliorated, inhibited orprevented, or its occurrence rate reduced, compared to that withoutanti-hANGPTL3 antibody treatment (e.g., ANGPTL3-mediated diseases ordisorders), by removing, inhibiting, reducing, or otherwise interferingwith, ANGPTL3 activity. Examples of diseases or disorders treatable bythe methods of the invention include, but are not limited to, thoseinvolving lipid metabolism, such as hyperlipidemia, hyperlipoproteinemiaand dyslipidemia, including atherogenic dyslipidemia, diabeticdyslipidemia, hypertriglyceridemia, including severehypertriglyceridemia with TG>1000 mg/dL, hypercholesterolemia,chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome,diabetes, etc.), lipodystrophy, lipoatrophy, and the like, which arecaused by, for example, decreased LPL activity and/or LPL deficiency,decreased LDL receptor (LDLR) activity and/or LDL receptor deficiency(e.g., homozygous familial hypercholesterolemia with LDLR^(−/−)),altered ApoC2, ApoE deficiency, increased ApoB, increased productionand/or decreased elimination of very low-density lipoprotein (VLDL),certain drug treatment (e.g., glucocorticoid treatment-induceddyslipidemia), any genetic predisposition, diet, life style, and thelike. The methods of the invention can also prevent or treat diseases ordisorders associated with or resulting from hyperlipidemia,hyper-lipoproteinemia, and/or dyslipidemia, including, but not limitedto, cardiovascular diseases or disorders, such as atherosclerosis,aneurysm, hypertension, angina, stroke, cerebrovascular diseases,congestive heart failure, coronary artery diseases, myocardialinfarction, peripheral vascular diseases, and the like; acutepancreatitis; nonalcoholic steatohepatitis (NASH); blood sugardisorders, such as diabetes; obesity, and the like.

Other examples of diseases or disorders treatable by the methods of theinvention include cancer/tumor as well as non-neoplasticangiogenesis-associated diseases or disorders, including ocularangiogenic diseases or disorders, such as age-related maculardegeneration, central retinal vein occlusion or branch retinal veinocclusion, diabetic retinopathy, retinopathy of prematurity, and thelike, inflammatory diseases or disorders, such as arthritis, rheumatoidarthritis (RA), psoriasis, and the like.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a sequence alignment of Peptides 1-3 used in ananti-hANGPTL3 antibody binding experiment (Example 5) against therelevant portions of hANGPTL3 sequence (i.e., within residues 30 to 70of SEQ ID NO:161 or GenBank #NP_(—)055310). Peptide 1 (control: ANGPTL4peptide; SEQ ID NO:168); Peptide 2 (ANGPTL3 peptide; SEQ ID NO:169); andPeptide 3 (ANGPTL3 peptide; SEQ ID NO:170).

FIG. 2 shows the results of anti-hANGPTL3 antibody binding to theN-terminal coiled-coil peptides of hANGPTL3 (Peptides 2 and 3) orhANGPTL4 (Peptide 1).

: Isotype Control; and ▪: H4H1276S antibody.

DETAILED DESCRIPTION

Before the present invention is described in detail, it is to beunderstood that this invention is not limited to particular methods, andexperimental conditions described, as such methods and conditions mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

DEFINITIONS

The term “human angiopoietin-like protein 3” or “hANGPTL3”, as usedherein, refers to ANGPTL3 having the nucleic acid sequence shown in SEQID NO:162 and the amino acid sequence of SEQ ID NO:161, or abiologically active fragment thereof.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region (HCVR)and a heavy chain constant region (C_(H); comprised of domains C_(H)1,C_(H)2 and C_(H)3). Each light chain is comprised of a light chainvariable region (LCVR) and a light chain constant region (C_(L)). TheHCVR and LCVR can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each HCVR and LCVR is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example, residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences.

The fully-human anti-hANGPTL3 antibodies disclosed herein may compriseone or more amino acid substitutions, insertions and/or deletions in theframework and/or CDR regions of the heavy and light chain variabledomains as compared to the corresponding germline sequences. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to germline sequences available from, forexample, public antibody sequence databases. The present inventionincludes antibodies, and antigen-binding fragments thereof, which arederived from any of the amino acid sequences disclosed herein, whereinone or more amino acids within one or more framework and/or CDR regionsare mutated to the corresponding residue(s) of the germline sequencefrom which the antibody was derived, or to the corresponding residue(s)of another human germline sequence, or to a conservative amino acidsubstitution of the corresponding germline residues(s) (such sequencechanges are referred to herein collectively as “germline mutations”). Aperson of ordinary skill in the art, starting with the heavy and lightchain variable region sequences disclosed herein, can easily producenumerous antibodies and antigen-binding fragments which comprise one ormore individual germline back-mutations or combinations thereof. Incertain embodiments, all of the framework and/or CDR residues within theV_(H) and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residues of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-ANGPTL3 antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-ANGPTL3 antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, 2 or 1, conservative amino acidsubstitution(s) relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein. In one embodiment, a HCVR comprises theamino acid sequence of SEQ ID NO:487 with 10 or fewer conservative aminoacid substitutions therein. In another embodiment, a HCVR comprises theamino acid sequence of SEQ ID NO:487 with 8 or fewer conservative aminoacid substitutions therein. In another embodiment, a HCVR comprises theamino acid sequence of SEQ ID NO:487 with 6 or fewer conservative aminoacid substitutions therein. In another embodiment, a HCVR comprises theamino acid sequence of SEQ ID NO:487 with 4 or fewer conservative aminoacid substitutions therein. In yet another embodiment, a HCVR comprisesthe amino acid sequence of SEQ ID NO:487 with 2 or 1 conservative aminoacid substitution(s) therein. In one embodiment, a LCVR comprises theamino acid sequence of SEQ ID NO:44 with 10 or fewer conservative aminoacid substitutions therein. In another embodiment, a LCVR comprises theamino acid sequence of SEQ ID NO:44 with 8 or fewer conservative aminoacid substitutions therein. In another embodiment, a LCVR comprises theamino acid sequence of SEQ ID NO:44 with 6 or fewer conservative aminoacid substitutions therein. In another embodiment, a LCVR comprises theamino acid sequence of SEQ ID NO:44 with 4 or fewer conservative aminoacid substitutions therein. In yet another embodiment, a LCVR comprisesthe amino acid sequence of SEQ ID NO:44 with 2 or 1 conservative aminoacid substitution(s) therein.

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and (optionally)constant domains. Such DNA is known and/or is readily available from,e.g., commercial sources, DNA libraries (including, e.g., phage-displayantibody libraries), or can be synthesized. The DNA may be sequenced andmanipulated chemically or by using molecular biology techniques, forexample, to arrange one or more variable and/or constant domains into asuitable configuration, or to introduce codons, create cysteineresidues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3—CDR3—FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (V)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (X) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (XII) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

In certain embodiments, antibody or antibody fragments of the inventionmay be conjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiological conditions. Specific binding canbe characterized by an equilibrium dissociation constant (K_(D)) ofabout 1×10⁻⁶ M or less (i.e., a smaller K_(D) denotes a tighterbinding). Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds hANGPTL3 may, however, exhibit cross-reactivityto other antigens, such as ANGPTL3 molecules from other species, forexample, cynomolgus monkey ANGPTL3, mouse ANGPTL3, rat ANGPTL3, and/orhANGPTL4 having the amino acid sequence of SEQ ID NO:164. Moreover,multi-specific antibodies (e.g., bispecifics) that bind to hANGPTL3 andone or more additional antigens are nonetheless considered antibodiesthat “specifically bind” hANGPTL3, as used herein.

The term “high affinity” antibody refers to those antibodies having abinding affinity to hANGPTL3, expressed as K_(D), of about 2×10⁻⁹ M orless, about 1.5×10⁻⁹ M or less, about 1×10⁻⁹ M or less, about 0.5×10⁻³ Mor less, about 0.25×10⁻⁶ M or less, about 1×10⁻¹⁰ M or less, or about0.5×10⁻¹⁰ M or less, as measured by surface plasmon resonance, e.g.,BIACORE™ or solution-affinity ELISA.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

By the term “slow off rate”, “Koff” or “k_(d)” is meant an antibody thatdissociates from hANGPTL3 with a rate constant of 4×10⁻³ s⁻¹ or less,3×10⁻³ s⁻¹ or less, 2×10⁻³ s⁻¹ or less, 1×10⁻³ s⁻¹ or less, 1×10⁻⁴s⁻¹ orless, as determined by surface plasmon resonance, e.g., BIACORE™.

By the term “intrinsic affinity constant” or “k_(a)” is meant anantibody that associates with hANGPTL3 at a rate constant of about 1×10³M⁻¹s⁻¹ or higher, as determined by surface plasmon resonance, e.g.,BIACORE™.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other mAbs having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hANGPTL3 is substantially free of mAbs that specifically bindantigens other than hANGPTL3). An isolated antibody that specificallybinds hANGPTL3 may, however, have cross-reactivity to other antigens,such as ANGPTL3 molecules from other species, such as cynomolgus monkey,mouse, rat, and/or other related proteins, such as human ANGPTL4.

A “neutralizing”, “blocking” or “abrogating” antibody, as used herein(or an antibody that “neutralizes”, “blocks” or “abrogates” ANGPTL3activity), is intended to refer to an antibody whose binding to ANGPTL3results in direct inhibition of at least one biological activity ofANGPTL3, as assessed by standard in vitro assays known in the art (forexample, see Examples below). The terms, “neutralize”, “inhibit”,“block” and “abrogate”, may be used herein interchangeably. A“non-blocking” antibody refers to an antibody whose binding to ANGPTL3does not directly block a targeted activity of ANGPTL3 as assessed bystandard in vitro assays, but yet may be an “interfering” antibody whosebinding to ANGPTL3 results in indirect inhibition, reduction,attenuation, or other interference, of at least one biological activityof ANGPTL3 in vivo, e.g., by enhancing the clearance of ANGPTL3 from thecirculation. Clearance of ANGPTL3 from the circulation can beparticularly enhanced by a combination of at least two non-blockingantibodies. The neutralization, inhibition, abrogation, reduction,attenuation or interference, of a biological activity of ANGPTL3 can beassessed by measuring one or more indicators of ANGPTL3 biologicalactivity by one or more of several standard in vitro or in vivo assaysknown in the art (also see Examples below).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positionswhich are not identical differ by conservative amino acid substitutions.A “conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent or degree of similarity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids that have side chains withsimilar chemical properties include 1) aliphatic side chains: glycine,alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl sidechains: serine and threonine; 3) amide-containing side chains:asparagine and glutamine; 4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, andhistidine; 6) acidic side chains: aspartate and glutamate, and 7)sulfur-containing side chains: cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443 45. A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and (1997)Nucleic Acids Res. 25:3389 402.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, the age and the sizeof a subject treated, the route of administration, and the like, andwill be ascertainable by one skilled in the art using known techniques(see, for example, Lloyd (1999) The Art, Science and Technology ofPharmaceutical Compounding).

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind toANGPTL3.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to ANGPTL3 areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, and the like.

In general, the antibodies of the instant invention possess highaffinities, typically possessing K_(D) of from about 10⁻¹² M throughabout 10⁻⁹ M, when measured by binding to antigen either immobilized onsolid phase or in solution phase. The mouse constant regions arereplaced with desired human constant regions, for example, wild-typeIgG1 or IgG4, or modified IgG1 or IgG4, to generate the fully humanantibodies of the invention. While the constant region selected may varyaccording to specific use, high affinity antigen-binding and targetspecificity characteristics of the antibodies reside in the variableregion.

Epitope Mapping and Related Technologies

To screen for antibodies that bind to a particular epitope, a routinecross-blocking assay such as that described in Antibodies, Harlow andLane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed.Other methods include alanine scanning mutants, peptide blots (Reineke(2004) Methods Mol Biol 248:443-63), or peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer (2000) ProteinScience 9: 487-496).

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (US2004/0101920). Each category may reflect a unique epitope eitherdistinctly different from or partially overlapping with epitoperepresented by another category. This technology allows rapid filteringof genetically identical mAbs, such that characterization can be focusedon genetically distinct mAbs. When applied to hybridoma screening, MAPmay facilitate identification of rare hybridoma clones that produce mAbshaving the desired characteristics. MAP may be used to sort theanti-ANGPTL3 mAbs of the invention into groups of mAbs binding differentepitopes.

ANGPTL3 contains an amino-terminal coiled-coil domain and acarboxyl-terminal fibrinogen like domain (FD) and the ANGPTL3 proteinforms an oligomer in the absence of intermolecular disulfide bonds (Geet al., 2005, J Lipid Res 46:1484-1490). It has been reported that theN-terminal coiled-coil domain is important in the inhibition of LPLactivity (Ono et al., 2003, J Biol Chem 278:41804-41809). Thus, incertain embodiments, the anti-hANGPTL3 antibody or antigen-bindingfragment of an antibody binds an epitope within the N-terminalcoiled-coil domain (residues 17-209) of hANGPTL3 (SEQ ID NO:161) andneutralizes at least one activity of hANGPTL3 (e.g., inhibition of LPLactivity). In another embodiments, the anti-hANGPTL3 antibody orantigen-binding fragment thereof binds an epitope within the N-terminalcoiled-coil domain of hANGPTL3 and neutralizes at least one activity ofhANGPTL3, with the proviso that the antibody or fragment thereof doesnot bind to the ANGPTL3 peptide of SEQ ID NO:170. In one embodiment, theantibody or fragment thereof specifically binds an epitope withinresidues 17 to 200, 17 to 100, 17 to 70, 17 to 65, 17 to 60, 17 to 57,17 to 55, 17 to 50, 17 to 45, 17 to 40, or 17 to 35, of hANGPTL3 (SEQ IDNO:161), optionally with the proviso that the antibody or fragmentthereof does not bind to the ANGPTL3 peptide of SEQ ID NO:170. Inanother embodiment, the antibody or fragment thereof specifically bindsan epitope within residues 40 to 200, 40 to 100, 40 to 70, 50 to 200, 50to 100, 50 to 70, 58 to 200, 58 to 100, 58 to 70, 58 to 68, or 61 to 66(known as a “heparin-binding motif”) of hANGPTL3 (SEQ ID NO:161),optionally with the proviso that the antibody or fragment thereof doesnot bind to the ANGPTL3 peptide of SEQ ID NO:170. In some embodiments,the antibody or antibody fragment binds an epitope which may involvemore than one of the enumerated epitopes or residues within theN-terminal coiled-coil region of hANGPTL3, optionally with the provisothat the antibody or fragment thereof does not bind to the ANGPTL3peptide of SEQ ID NO:170.

In other embodiments, hANGPTL3 antibody or fragment thereof binds one ormore fragments of hANGPTL3, for example, a fragment of at least 5residues, at least 7 residues, at least 10 residues, at least 20residues, at least 30 residues, at least 50 residues, at least 70residues, at least 100 residues, at least 150 residues, or at least 200residues, of hANGPTL3 (SEQ ID NO:161), optionally with the proviso thatthe antibody or fragment thereof does not bind to the ANGPTL3 peptide ofSEQ ID NO:170.

The present invention includes hANGPTL3 antibodies that bind to the sameepitope as any of the specific exemplary antibodies described herein.Likewise, the present invention also includes anti-hANGPTL3 antibodiesthat compete for binding to hANGPTL3 or a hANGPTL3 fragment with any ofthe specific exemplary antibodies described herein.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-hANGPTL3 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-hANGPTL3antibody of the invention, the reference antibody is allowed to bind toa hANGPTL3 protein or peptide under saturating conditions. Next, theability of a test antibody to bind to the hANGPTL3 molecule is assessed.If the test antibody is able to bind to hANGPTL3 following saturationbinding with the reference anti-hANGPTL3 antibody, it can be concludedthat the test antibody binds to a different epitope than the referenceanti-hANGPTL3 antibody. On the other hand, if the test antibody is notable to bind to the hANGPTL3 molecule following saturation binding withthe reference anti-hANGPTL3 antibody, then the test antibody may bind tothe same epitope as the epitope bound by the reference anti-hANGPTL3antibody of the invention.

To determine if an antibody competes for binding with a referenceanti-hANGPTL3 antibody, the above-described binding methodology isperformed in two orientations: In a first orientation, the referenceantibody is allowed to bind to a hANGPTL3 molecule under saturatingconditions followed by assessment of binding of the test antibody to thehANGPTL3 molecule. In a second orientation, the test antibody is allowedto bind to a hANGPTL3 molecule under saturating conditions followed byassessment of binding of the reference antibody to the ANGPTL3 molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the ANGPTL3 molecule, then it is concluded thatthe test antibody and the reference antibody compete for binding tohANGPTL3. As will be appreciated by a person of ordinary skill in theart, an antibody that competes for binding with a reference antibody maynot necessarily bind to the identical epitope as the reference antibody,but may sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

Immunoconjugates

The invention encompasses a human anti-ANGPTL3 monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope. Cytotoxin agents include any agent that is detrimental tocells. Examples of suitable cytotoxin agents and chemotherapeutic agentsfor forming immunoconjugates are known in the art, see for example, WO05/103081.

Bispecifics

The antibodies of the present invention may be monospecific, bispecific,or multispecific. Multispecific mAbs may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al. (1991) J. Immunol. 147:60-69. The human anti-hANGPTL3 mAbs can belinked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment, to produce abispecific or a multispecific antibody with a second bindingspecificity.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Bioequivalents

The anti-hANGPTL3 antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described mAbs, but that retain the ability to bind humanANGPTL3. Such variant mAbs and antibody fragments comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described mAbs. Likewise, the anti-hANGPTL3antibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-hANGPTL3 antibody or antibody fragment that isessentially bioequivalent to an anti-hANGPTL3 antibody or antibodyfragment of the invention. Examples of such variant amino acid and DNAsequences are discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied. In one embodiment, two antigen-binding proteins arebioequivalent if there are no clinically meaningful differences in theirsafety, purity, and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-hANGPTL3 antibodies of the invention maybe constructed by, for example, making various substitutions of residuesor sequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-hANGPTL3 antibodies or antigen-binding fragments thereof of thepresent invention and the therapeutic methods using the same. Theadministration of therapeutic compositions in accordance with theinvention will be administered with suitable carriers, excipients, andother agents that are incorporated into formulations to provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, the purpose of the treatment, conditions,route of administration, and the like. When the antibody of the presentinvention is used for treating various conditions and diseases directlyor indirectly associated with ANGPTL3, including hypercholesterolemia,disorders associated with LDL and apolipoprotein B, and lipid metabolismdisorders, and the like, in an adult patient, it is advantageous tointravenously or subcutaneously administer the antibody of the presentinvention at a single dose of about 0.01 to about 20 mg/kg body weight,more preferably about 0.02 to about 7, about 0.03 to about 5, or about0.05 to about 3 mg/kg body weight. Depending on the severity of thecondition, the frequency and the duration of the treatment can beadjusted. In certain embodiments, the antibody or antigen-bindingfragment thereof of the invention can be administered as an initial doseof at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about5 to about 300 mg, or about 10 to about 200 mg, to about 100 mg, or toabout 50 mg. In certain embodiments, the initial dose may be followed byadministration of a second or a plurality of subsequent doses of theantibody or antigen-binding fragment thereof in an amount that can beapproximately the same or less than that of the initial dose, whereinthe subsequent doses are separated by at least 1 day to 3 days; at leastone week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 0.1 to about 800 mg per dosage form in aunit dose; especially in the form of injection, the aforesaid antibodyis contained in about 1 to about 500 mg, in about 5 to 300 mg, in about8 to 200 mg, and in about 10 to about 100 mg for the other dosage forms.

Combination Therapies

The invention further provides therapeutic methods for treating diseasesor disorders, which is directly or indirectly associated with hANGPTL3,by administering a hANGPTL3 antibody or fragment thereof of theinvention in combination with one or more additional therapeutic agents.The additional therapeutic agent may be one or more of any agent that isadvantageously combined with one or more antibodies or fragments thereofof the invention, including HMG-CoA reductase inhibitors, such ascerovastatin, atorvastatin, simvastatin, pitavastin, ros uvastatin,fluvastatin, lovastatin, pravastatin, and the like; niacin; variousfibrates, such as fenofibrate, bezafibrate, ciprofibrate, clofibrate,gemfibrozil, and the like; LXR transcription factor activators, and thelike. Furthermore, the hANGPTL3 antibody or fragment thereof of theinvention can be co-administered with other ANGPTL3 inhibitors as wellas inhibitors of other molecules, such as ANGPTL4, ANGPTL5, ANGPTL6 andproprotein convertase subtilisin/kexin type 9 (PCSK9), which areinvolved in lipid metabolism, in particular, cholesterol and/ortriglyceride homeostasis. Inhibitors of these molecules include smallmolecules and antibodies that specifically bind to these molecules andblock their activity (see, for example, anti-PCSK9 antibodies disclosedin U.S. 2010/0166768 A1).

Furthermore, the additional therapeutic agent may be one or moreanti-cancer agents, such as chemotherapeutic agents, anti-angiogenicagents, growth inhibitory agents, cytotoxic agents, apoptotic agents,and other agents well known in the art to treat cancer or otherproliferative diseases or disorders. Examples of anti-cancer agentsinclude, but are not limited to, an anti-mitotic agent, such asdocetaxel, paclitaxel, and the like; a platinum-based chemotherapeuticcompound, such as cisplatin, carboplatin, iproplatin, oxaliplatin, andthe like; or other conventional cytotoxic agent, such as 5-fluorouracil,capecitabine, irinotecan, leucovorin, gemcitabine, and the like, andanti-angiogenic agents, including vascular endothelial growth factor(VEGF) antagonists, such as anti-VEGF antibodies, e.g., bevacizumab(AVASTIN®, Genentech) and a receptor-based blocker of VEGF, e.g., “VEGFtrap” described in U.S. Pat. No. 7,070,959, delta-like ligand 4 (DII4)antagonists, such as anti-DII4 antibodies as described in U.S. PatentApplication Publication No. 2008/0181899, and a fusion proteincontaining the extracellular domain of DII4, e.g., DII4-Fc as describedin U.S. Patent Application Publication No. 2008/0107648; inhibitors ofreceptor tyrosine kinases and/or angiogenesis, including sorafenib(NEXAVAR® by Bayer Pharmaceuticals Corp.), sunitinib (SUTENT® byPfizer), pazopanib (VOTRIENT™ by GlaxoSmithKline), toceranib (PALLADIA™by Pfizer), vandetanib (ZACTIMA™ by AstraZeneca), cediranib (RECENTIN®by AstraZeneca), regorafenib (BAY 73-4506 by Bayer), axitinib (AG013736by Pfizer), lestaurtinib (CEP-701 by Cephalon), erlotinib (TARCEVA® byGenentech), gefitinib (IRESSA™ by AstraZeneca), BIBW 2992 (TOVOK™ byBoehringer Ingelheim), lapatinib (TYKERB® by GlaxoSmithKline), neratinib(HKI-272 by Wyeth/Pfizer), and the like, and pharmaceutically acceptablesalts, acids or derivatives of any of the above. In addition, othertherapeutic agents, such as analgesics, anti-inflammatory agents,including non-steroidal anti-inflammatory drugs (NSAIDS), such as Cox-2inhibitors, and the like, may be also co-administered with the hANGPTL3antibody or fragment thereof of the invention so as to ameliorate and/orreduce the symptoms accompanying the underlying cancer/tumor.

The hANGPTL3 antibody or fragment thereof of the invention and theadditional therapeutic agent(s) can be co-administered together orseparately. Where separate dosage formulations are used, the antibody orfragment thereof of the invention and the additional agents can beadministered concurrently, or separately at staggered times, i.e.,sequentially, in appropriate orders.

Diagnostic Uses of the Antibodies

The anti-ANGPTL3 antibodies of the present invention can be also used todetect and/or measure ANGPTL3 in a sample, e.g., for diagnosticpurposes. For example, an anti-ANGPTL3 Ab or fragment thereof, can beused to diagnose a condition or disease characterized by aberrantexpression (e.g., over-expression, under-expression, lack of expression,etc.) of ANGPTL3. Exemplary diagnostic assays for ANGPTL3 may comprise,e.g., contacting a sample obtained from a patient, with an anti-ANGPTL3Ab of the invention, wherein the anti-ANGPTL3 antibody is labeled with adetectable label or reporter molecule or used to selectively capture andisolate ANGPTL3 protein from patient samples. Alternatively, anunlabeled anti-ANGPTL3 Ab can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹³¹I or ¹²⁵I; a fluorescent orchemiluminescent moiety, such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase,horseradish peroxidase, or luciferase. Assays that can be used to detector measure ANGPTL3 in a sample include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting(FACS), and the like.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human ANGPTL3

VELOCIMMUNE™ mice were immunized with human ANGPTL3, and the antibodyimmune response monitored by antigen-specific immunoassay using serumobtained from these mice. Anti-hANGPTL3 expressing B cells wereharvested from the spleens of immunized mice shown to have elevatedanti-hANGPTL3 antibody titers and were fused with mouse myeloma cells toform hybridomas. The hybridomas were screened and selected to identifycell lines expressing hANGPTL3-specific antibodies using assays asdescribed below. The assays identified several cell lines that producedchimeric anti-hANGPTL3 antibodies, e.g., H1M896N.

Human ANGPTL3-specific antibodies were also isolated directly fromantigen-immunized B cells without fusion to myeloma cells, as describedin U.S. 2007/0280945 A1. Heavy and light chain variable regions werecloned to generate fully human anti-hANGPTL3 antibodies of IgG4 isotypedesignated as H4H1248P, H4H1250P, H4H1263S, H4H1268S, H4H1276S,H4H1279P, H4H1282P, H4H1292P, H4H1295P and H4H1296P. Stable recombinantantibody-expressing CHO cell lines were established.

Example 2 Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage was identified for each Heavy Chain VariableRegion (HCVR) and Light Chain Variable Region (LCVR). Table 1 shows thegene usage for selected antibodies in accordance with the invention.

TABLE 1 HCVR LCVR Antibody V_(H) D_(H) J_(H) V_(K) J_(K) H4H1248P 3-30 1-26 6 1-12 3 H4H1250P 3-30 1-7 6 1-5  1 H4H1263S 3-30  3-10 6 1-12 3H4H1268S 6-1  6-6 4 1-5  1 H4H1276S 3-43 3-3 3 1-5  2 H4H1279P 3-11 1-14 1-39 4 H4H1282P 1-18  3-10 4 1-9  4 H4H1292P 3-11 1-1 4 1-39 4H4H1295P 1-18  6-25 4 2-30 2 H4H1296P 3-11 1-1 4 1-39 4 H1M896N 3-23 3-10 4 1-5  1

Table 2 shows the heavy and light chain variable region amino acidsequence pairs of selected anti-hANGPTL3 antibodies and theircorresponding antibody identifiers. The N, P and S designations refer toantibodies having heavy and light chains with identical CDR sequencesbut with sequence variations in regions that fall outside of the CDRsequences (i.e., in the framework regions). Thus, N, P and S variants ofa particular antibody have identical CDR sequences within their heavyand light chain variable regions but contain modifications within theframework regions.

TABLE 2 HCVR/LCVR HCVR/LCVR Name SEQ ID NOs Name SEQ ID NOs H4H1248P 2/10 H4H1279P 82/90 H4H1250P 18/26 H4H1282P  98/106 H4H1263S 34/42H4H1292P 114/122 H4H1268S 50/58 H4H1295P 130/138 H4H1276S 66/74 H4H1296P146/154 H1M896N 180/188 — —

Example 3 Kinetic Parameters of Anti-hANGPTL3 Antibodies Binding toANGPTL3

All kinetic binding experiments were performed at 25° C. or 37° C. on aBIACORE™ T200 label-free molecular interaction instrument (GEHealthcare) using a CM5 sensor chip. Briefly, an antigen capture surfacewas generated by covalently coupling either an anti-mouse IgG-specificantibody (anti-mFc; GE Healthcare; catalog #BR-1008-38) or ananti-pentahistidine-specific antibody (Qiagen; catalog #34660) to thesurface of a CM5 sensor chip using a standard amine coupling method.Using HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20,pH 7.4) or PBSP (10 mM sodium phosphate, 2.7 mM KCl, 137 mM NaCl, 0.025%Surfactant P20, pH 7.2 or 5.75) as the running buffer, human and speciesvariants of ANGPTL3 with oligohistidine tags were captured on theanti-penia-histidine coupled surface until a binding response of4.4-46.5 RUs was achieved. The captured recombinant proteins were:full-length mature human ANGPTL3 (i.e., amino acid residues 17-460 ofSEQ ID NO:161) with a C-terminal decahistidine tag[hANGPTL3(17-460)-His; R&D Systems, MN; catalog #3829-AN], N-terminalcoiled-coil domain of hANGPTL3 (i.e., amino acid residues 17-170 of SEQID NO:161) containing a C-terminal hexahistidine tag[hANGPTL3(17-170)-His], N-terminal coiled-coil domain of ANGPTL3 fromMacaca fascicularis [i.e., amino acid residues 17-170 of SEQ ID NO:177(a partial sequence of Macaca fascicularis ANGPTL3)] containing amyc-myc-hexahistidine tag [MfANGPTL3(17-170)-mmH; SEQ ID NO:167],full-length mature ANGPTL3 from Mus musculus (i.e., amino acid residues17-455 of SEQ ID NO:163) with a C-terminal decahistidine tag[mANGPTL3(17-455)-His; R&D Systems, MN; catalog #136-AN], N-terminalcoiled-coil domain of ANGPTL3 from Mus musculus (i.e., amino acidresidues 17-240 of SEQ ID NO:163) containing a hexahistidine tag[mANGPTL3(17-240)-His; SEQ ID NO:166], and N-terminal coiled-coil domainof ANGPTL3 from Rattus norvegicus (i.e., amino acid residues 17-240 ofSEQ ID NO:175) containing a myc-myc-hexahistidine tag[rANGPTL3(17-240)-mmH; SEQ ID NO:176]. In addition, the N-terminalcoiled-coil domain of hANGPTL3 (i.e., amino acid residues 17-169 of SEQID NO:161) containing a C-terminal mouse Fc fusion[hANGPTL3(17-169)-mFc; SEQ ID NO:165] was captured on the anti-mFccoupled surface until a binding response of 24.8±1.5 RUs was achieved.To measure association and dissociation rates for formation of theantibody/antigen complex, a single (Tables 3 and 7) or multiple (Tables4-6) concentrations of antibody were injected across the capturedprotein surface at a flow rate of 50 μl/minute for 3 minutes anddissociation of the complex was monitored for 20 minutes. Binding datawere processed and fitted to a 1:1 binding model with mass transportusing Scrubber version 2.0a (BioLogic Software). The kinetic half-lives(t_(1/2)) were calculated from the dissociation rate constant, kd.

Table 3 shows the binding of various anti-ANGPTL3 antibodies to hANGPTL3at 25° C., pH 7.4, in HBS-EP buffer.

TABLE 3 Ab Clones Protein ka (M⁻¹s⁻¹) kd (s⁻¹) K_(D) (nM) t_(1/2) (min)H4H1248P hANGPTL3(17-169)-mFc 4.57E+05 2.72E−03 5.95 4hANGPTL3(17-460)-His 4.40E+05 2.47E−03 5.62 5 H4H1250PhANGPTL3(17-169)-mFc 1.25E+06 6.51E−04 0.519 18 hANGPTL3(17-460)-His9.04E+05 6.57E−04 0.726 18 H4H1263S hANGPTL3(17-169)-mFc 6.77E+054.22E−03 6.23 3 hANGPTL3(17-460)-His 5.08E+05 1.26E−03 2.47 9 H4H1268ShANGPTL3(17-169)-mFc 1.16E+06 8.35E−04 0.721 14 hANGPTL3(17-460)-His1.29E+06 1.89E−03 1.47 6 H4H1276S hANGPTL3(17-169)-mFc 5.82E+05 3.83E−040.659 30 hANGPTL3(17-460)-His 3.44E+05 4.64E−04 1.35 25 H4H1279PhANGPTL3(17-169)-mFc 6.58E+05 5.53E−06 0.00841 2088 hANGPTL3(17-460)-His2.88E+05 1.14E−04 0.394 102 H4H1282P hANGPTL3(17-169)-mFc 1.28E+065.92E−05 0.0463 195 hANGPTL3(17-460)-His 9.57E+05 9.26E−05 0.0968 125H4H1292P hANGPTL3(17-169)-mFc 6.86E+05 1.77E−04 0.257 65hANGPTL3(17-460)-His 3.41E+05 2.48E−04 0.727 47 H4H1295PhANGPTL3(17-169)-mFc 3.52E+05 7.95E−05 0.226 145 hANGPTL3(17-460)-His3.73E+05 7.35E−05 0.197 157 H4H1296P hANGPTL3(17-169)-mFc 6.41E+053.92E−05 0.0611 295 hANGPTL3(17-460)-His 3.01E+05 4.12E−05 0.137 280

As shown in Table 3, the anti-hANGPLT3 antibodies bound to thefull-length protein with a C-terminal decahistidine tag[hANGPTL3(17-460)-His] with calculated equilibrium dissociationconstants (K_(D)=kdlka) ranging from 96.8 pM to 5.62 nM and to theN-terminal coiled-coil domain with a C-terminal Fc fusion[hANGPTL3(17-169)-mFc] with K_(D)s ranging from 8.41 pM to 6.23 nM.

Tables 4 and 5 show the cross-species binding of H4H1276S to ANGPTL3 at25° C. and 37° C., respectively, at pH 7.4, in HBS-EP buffer. Table 6shows the binding of H4H1276S to human and cynomolgus ANGPTL3, at 25° C.or 37° C., at pH 5.75 or pH 7.2, in PBSP buffer.

TABLE 4 25° C. Ab Clone Protein ka (M⁻¹s⁻¹) kd (s⁻¹) K_(D) (nM) t_(1/2)(min) H4H1276S hANGPTL3(17-170)-His 9.73E+05 9.12E−04 0.938 12.7hANGPTL3(17-460)-His 5.88E+05 2.89E−04 0.491 40.0 MfANGPTL3(17-170)-mmH1.35E+06 5.35E−04 0.396 21.6 mANGPTL3(17-240)-His 6.70E+05 3.07E−040.458 37.6 mANGPTL3(17-455)-His 1.29E+06 3.46E−04 0.268 33.4rANGPTL3(17-240)-mmH 1.35E+06 7.18E−04 0.530 16.1

TABLE 5 37° C. Ab Clone Protein ka (M⁻¹s⁻¹) kd (s⁻¹) K_(D) (nM) t_(1/2)(min) H4H1276S hANGPTL3(17-170)-His 1.59E+06 2.41E−03 1.52 4.8hANGPTL3(17-460)-His 6.32E+05 8.12E−04 1.29 14.2 MfANGPTL3(17-170)-mmH1.87E+06 1.17E−03 0.625 9.9 mANGPTL3(17-240)-His 8.19E+05 9.64E−04 1.1812.0 mANGPTL3(17-455)-His 1.94E+06 7.91E−04 0.408 14.6rANGPTL3(17-240)-mmH 2.05E+06 1.93E−03 0.940 6.0

TABLE 6 Ab Clone Protein ka (M⁻¹s⁻¹) kd (s⁻¹) K_(D) (nM) t_(1/2) (min)H4H1276S hANGPTL3(17-170)-His 1.00E+06 1.10E−03 1.09 10.5 pH 7.2hANGPTL3(17-460)-His 5.99E+05 4.02E−04 0.670 28.8 25° C.MfANGPTL3(17-170)-mmH 1.45E+06 5.38E−04 0.370 21.5 H4H1276ShANGPTL3(17-170)-His 2.80E+05 6.72E−03 24.0 1.7 pH 5.75hANGPTL3(17-460)-His 7.32E+04 4.94E−03 67.5 2.3 25° C.MfANGPTL3(17-170)-mmH 2.06E+05 4.32E−03 21.0 2.7 H4H1276ShANGPTL3(17-170)-His 1.57E+06 2.73E−03 1.74 4.2 pH 7.2hANGPTL3(17-460)-His 6.67E+05 1.18E−03 1.76 9.8 37° C.MfANGPTL3(17-170)-mmH 1.94E+06 1.36E−03 0.700 8.5 H4H1276ShANGPTL3(17-170)-His 1.22E+06 3.24E−02 26.7 0.4 pH 5.75hANGPTL3(17-460)-His 4.71E+04 1.07E−02 227 1.1 37° C.MfANGPTL3(17-170)-mmH 2.78E+05 5.21E−03 18.8 2.2

As shown in Tables 4-6, antibody H4H1276S exhibited binding to ANGPTL3from monkey, mouse, and rat with binding affinities and kineticconstants similar to those for binding to human ANGPTL3.

Table 7 shows the binding of selected anti-ANGPTL3 antibodies tohANGPTL3 and mANGPTL3 at 37° C., pH 7.4, in HBS-EP buffer. NB: Notbound.

TABLE 7 Ab Clones Protein ka (M⁻¹s⁻¹) kd (s⁻¹) K_(D) (nM) t_(1/2) (min)H1M896N hANGPTL3(17-460)-His 3.36E+06 5.30E−04 1.58E−10 22mANGPTL3(17-455)-His 3.62E+06 2.47E−03 6.82E−10 5 H4H1248PhANGPTL3(17-460)-His 1.96E+06 1.93E−03 9.86E−10 6 mANGPTL3(17-455)-HisNB NB NB NB H4H1250P hANGPTL3(17-460)-His 3.50E+06 1.13E−03 3.24E−10 10mANGPTL3(17-455)-His 3.18E+06 1.55E−03 4.86E−10 7 H4H1263ShANGPTL3(17-460)-His 4.74E+06 1.81E−03 3.81E−10 6 mANGPTL3(17-455)-HisNB NB NB NB H4H1279P hANGPTL3(17-460)-His 4.74E+06 1.81E−03 3.81E−10 6mANGPTL3(17-455)-His 2.15E+06 3.59E−04 1.67E−10 32 H4H1292PhANGPTL3(17-460)-His 1.89E+06 1.94E−03 1.02E−09 6 mANGPTL3(17-455)-His3.92E+06 1.49E−03 3.80E−10 8

As shown in Table 7, the anti-hANGPLT3 antibodies bound to thefull-length protein with a C-terminal decahistidine tag[hANGPTL3(17-460)-His] at pH 7.4 and 37° C. with calculated equilibriumdissociation constants (K_(D)=kd/ka) ranging from 158 pM to 1.02 nM andto mouse ANGPTL3 [mANGPTL3(17-455)-His] with K_(o)s ranging from 167 pMto 682 pM, except for H4H1248P and H4H1263S, which did not showdetectable binding to mANGPTL3. Also shown are the kinetic half-lives(t_(1/2)).

Example 4 Biacore Cross-Competition Study for Anti-ANGPTL3 Antibodies

Cross competition experiments were performed at 25° C. on a Biacoreessentially as described in Example 3 above. Briefly, using HBS-EP asthe running buffer, full-length hANGPTL3 (i.e., amino acid residues17-460 of SEQ ID NO:161) with a C-terminal decahistidine tag[hANGPTL3(17-460)-His; R&D Systems, MN; catalog #3829-AN] was capturedon the anti-penta-histidine coupled surface until a binding response of64 RUs was achieved. To determine whether two antibodies could bindsimultaneously to the captured ANGPTL3, antibody pairs were injectedsequentially, each at 167 nM at a flow rate of 4 μl/minute for 15minutes, over the surface, and the maximum binding response signal (RU)was measured for each binding event. The results are shown in Table 8with binding response for the first antibody (mAb1), followed by bindingresponse of the second antibody (mAb2) on the ANGPTL3 surface pre-loadedwith the first antibody. Numbers in bold indicate that the antibodypairs are able to bind to hANGPTL3 simultaneously. Numbers in italicsindicate that that the antibody pairs are able to bind to hANGPTL3simultaneously when added sequentially in one direction but not theother. Brackets indicate self-self competition.

TABLE 8 hANGPTL3(17- 25 μg/ml 460)-His mAb1 binding 25 μg/ml mAb2binding response (RU) Ab Clones captured (RU) response (RU) H1M896NH4H1250P H4H1279P H4H1292P H1M896N 64 ± 4 171 ± 1.8   [−4]  −4 47 50H4H1250P 210 ± 8.3   −4  [13]  9  8 H4H1279P 45 ± 1.8 186 213 [−3] −1H4H1292P 48 ± 0.7 182 220 −3 [−2] Negative 81 ± 1.8 149 187 47 48Control

As shown in Table 8, antibody pairs H1M896N/H4H1279P andH1M896N/H4H1292P were able to bind simultaneously to immobilizedANGPTL3, regardless of the order of addition of the antibodies. H4H1250Pbound to ANGPTL3 pre-bound with H4H1279P; however, when the order ofantibody addition was reversed, H4H1279P exhibited a binding signalapproximately 24% of the expected maximal response after ANGPTL3 waspre-bound with H4H1250P. Similarly, H4H1250P bound to ANGPTL3 pre-boundwith H4H1292P; however, when the order of antibody addition wasreversed, H4H1292P exhibited a binding signal approximately 20% of theexpected maximal response after ANGPTL3 was pre-bound with H4H1250P.

Example 5 Anti-hANGPTL3 Antibody Binding to ANGPTL3 N-TerminalCoiled-Coil Peptides

To assess the binding of the anti-ANGPTL3 antibody H4H1276S to peptidesderived from the N-terminal coiled-coil region of ANGPTL3, a label-freebiosensor binding assay was performed using OCTET® RED system (ForteBio,Inc.). For immobilization onto the sensor, peptides were labeled witheither an N-terminal biotin tag [separated by a flexible linker, aminoacids “AGSSPGG” (SEQ ID NO:171), for Peptide 1 and Peptide 2; and aminoacids “GGGGS” (SEQ ID NO:172) for Peptide 3], or a C-terminal biotin tag[separated by a flexible linker, amino acids “GPSSGAPPPK” (SEQ IDNO:173), for Peptide 1 and Peptide 2; and amino acids “GGGGSK” (SEQ IDNO:174) for Peptide 3]. The peptide sequences tested were: A negativecontrol peptide, N-terminal biotin tagged Peptide1 (SEQ ID NO:168;residues Arg34 to Leu66 of human ANTGPTL4 of SEQ ID NO:164); andpeptides derived from the N-terminal coiled-coil region of ANGPTL3,i.e., N-terminal biotin tagged Peptide 2 (SEQ ID NO:169; residues Arg36to Leu68 of hANGPTL3 of SEQ ID NO:161); C-terminal biotin tagged Peptide2; N-terminal biotin tagged Peptide 3 (SEQ ID NO:170; corresponds toresidues Glu32 to Leu57 of hANGPTL3 of SEQ ID NO:161); and C-terminalbiotin tagged Peptide 3. Peptide sequences are also shown in FIG. 1.Streptavidin-coated biosensor tips were coated with the biotinylatedpeptides resulting in 1.22-2.26 nm of binding response units dependingon the peptide. The peptide-coated biosensor tips were then dipped intowells containing 1 μM of either anti-ANGPTL3 antibody H4H1276S or anisotype-matched negative control antibody, and binding was monitored for2.5 minutes. The binding response of H4H1276S and the isotype controlantibody to each of the peptides is summarized in FIG. 2. It wasobserved that H4H1276S binds to the ANGPTL3 linear sequence defined byPeptide 2 but not the overlapping but distinct sequence defined byPeptide 3 (see also FIG. 1). The isotype control antibody also served asa positive control for loading of Peptide 1 hANGPTL4 peptide) onto thebiosensor, because this isotype control antibody specifically recognizeshANGPTL4. As shown in FIG. 2, the binding of the control antibody toPeptide 1 confirmed that Peptide 1 was present on the sensor surface andso were the other peptides.

Example 6 Inhibition of hANGPTL3 by Anti-hANGPTL3 Antibodies in LPLBioassays

Lipoprotein Lipase (LPL) plays a critical role in lipid metabolism inhumans. LPL catalyzes hydrolysis of triglycerides and releases fattyacids to be metabolized. ANGPTL3 inhibits LPL activity leading toincreased level of lipids (Oike et al., 2005, Trends in MolecularMedicine 11(10):473-479). The N-terminal coiled-coil region of ANGPTL3inhibits LPL when expressed without the C-terminal fibrinogen region andtherefore appears to confer its inhibitory function. A cell-freebioassay was developed to determine the ability of anti-ANGPTL3antibodies to inhibit ANGPTL3-induced decrease in LPL activity.

Inhibition of hANGPTL3 activity by anti-ANGPTL3 antibodies wasdetermined using the CONFLUOLIP™ Continuous Fluorometric Lipase Test(Progen, Germany) using three hANGPTL3 proteins: full-length maturehANGPTL3 (i.e., amino acid residues 17-460 of SEQ ID NO:161) with aC-terminal decahistidine tag [hANGPTL3(17-460)-His; R&D Systems, MN;catalog #3829-AN], the N-terminal coiled-coil region (i.e., amino acidresidues 17-169 of SEQ ID NO:161) with a C-terminal mouse Fc fusion[hANGPTL3(17-169)-mFc; SEQ ID NO:165], and the N-terminal coiled-coildomain of hANGPTL3 (i.e., amino acid residues 17-170 of SEQ ID NO:161)containing a C-terminal hexahistidine tag [hANGPTL3(17-170)-His].

Briefly, bovine LPL (final concentration of 2 nM), human ApoCII (acofactor of LPL, final concentration of 0.23 μM), and BSA (finalconcentration of 2 mg/mL) in PBS were premixed. The hANGPTL3 recombinantproteins were added to the Apo/LPL mixture (final concentrations of80-100 nM). The Apo/LPL/ANGPTL3 protein mixtures were then addedtogether with serially diluted anti-hANGPTL3 antibodies and incubated atroom temperature for 30 minutes. Following the incubation, 100 μl ofreconstituted lipase substrate,1-trinitrophenyl-amino-dodecanoyl-2-pyrendecanoyl-3-O-hexadecyl-sn-glycerol(LS-A, Progen), was added to 25 μl of the antibody mixture to a 96-wellassay plate and incubated at 37° C. for two hours. Fluorescence was thenmeasured at 342 nm/400 nm (excitation/emission) using a FLEXSTATION® 3Microplate Reader (Molecular Devices, CA). Fluorescence is directlyproportional to LPL activity.

Antibody H4H1276S exhibited inhibition of hANGPTL3's inhibitory activityagainst LPL. A full dose-response using the hANGPTL3 protein in the LPLassay was first performed to determine the ANGPTL3 EC₅₀ for eachexperiment, and IC₅₀ determinations for the antibody were then performedusing constant concentrations of ANGPTL3 protein, as shown in Table 8.The antibody concentrations required for 50% maximum inhibition (IC₅₀)was determined to be 9.6 nM for 80 nM hANGPTL3(17-460)-His, 2.9 nM for100 nM hANGPTL3(17-170)-His and 21 nM for 80 nM hANGPTL3(17-169)-mFc,respectively. Antibody concentrations ranged from 0 to 300 nM fortesting human ANGPTL3 proteins.

Similarly, H4H1276S was tested in the LPL bioassay for its ability toinhibit cross-species orthologs: the cynomolgus monkey N-terminal region(amino acid residues 17-170 of SEQ ID NO:177) expressed with anC-terminal myc-myc-hexa-histidine tag [MfANGPTL3(17-170)-mmH; SEQ IDNO:167], the mouse ortholog N-terminal region amino acid residues 17-240of SEQ ID NO:163 with a C-terminal hexa-histidine tag[mANGPTL3(17-240)-His; SEQ ID NO:166], and full-length mature ANGPTL3from Mus musculus (i.e., amino acid residues 17-455 of SEQ ID NO:163)with a C-terminal decahistidine tag [mANGPTL3(17-455)-His; R&D Systems,MN; catalog #136-AN]. IC50s were determined to be 10 nM for 500 nMconstant MfANGPTL3(17-170)-mmH, 14 nM for 80 nM constantmANGPTL3(17-455)-His, and 31 nM for 500 nM constantmANGPTL3(17-240)-His. Antibody concentrations ranged from 0 to 600 nMfor testing monkey and mouse ANGPTL3 proteins. The results aresummarized in Table 9.

Antibodies against the N-terminal region of the homologous proteinANGPTL4 have also been shown to block the inhibitory function of ANGPTL4on LPL (Lee et al., 2009, J. Biol. Chem. 284:13735-13745). Therefore, toevaluate possible cross-reactivity to ANGPTL4, the inhibitoryanti-ANGPTL3 antibody H4H1276S was also tested against human ANGPTL4 inthe LPL lipase assay, conducted as described above for the ANGPTL3proteins. A recombinant form of the coiled-coil region of human ANGPTL4(residues 26-148 of SEQ ID NO:164) with a C-terminal mouse IgG2a Fcfusion [hANGPTL4(26-148)-mFc, SEQ ID NO:178] exhibited an EC50 in theLPL assay of 0.2 nM (Table 9). H4H1276S, tested through a concentrationrange of 0-600 nM, did not block this inhibition (NB: Not bound; inTable 9).

TABLE 9 Human Human Human Monkey Mouse Mouse Human ANGPTL3 ANGPTL3ANGPTL3 ANGPTL3 ANGPTL3 ANGPTL3 ANGPTL4 (17-460)-His (17-170)-His(17-169)-mFc (17-170)-mmH (17-455)-His (17-240)-His (26-148)-mFc EC₅₀(nM) 50 91 16 625 33 199 0.2 Constant 80 100 80 500 80 500 2 ANGPTL3 or4 (nM) IC₅₀ H4H 1276S 9.6 2.9 21 10 14 31 NB (nM) IgG4 cont. NB NB NB NBNB NB NB

As shown above, H4H1276S inhibited human ANGPTL3 (full-length andN-terminal), monkey ANGPTL3 (N-terminal) protein and mouse ANGPTL3(full-length and N-terminal) activity at comparable degrees with an IC₅₀range of about 3-31 nM.

A subset of antibodies were also tested to determine if combinations oftwo ANGPTL3 non-blocking antibodies added simultaneously could block theLPL inhibitory activity of ANGPTL3. Pairs of antibodies were tested forinhibiting the N-terminal domains of both human and mouse ANGPTL3, i.e.,hANGPTL3(17-169)-mFc and mANGPTL3(17-240)-His, respectively. For thisassay, the ANGPTL3 proteins exhibited IC₅₀ values for blocking LPL of 47nM [for hANGPTL3(17-169)-mFc] and 341 nM [for mANGPTL3(17-240)-His]. Thefollowing pairs, when added at final concentrations for each antibody ofat least 200 nM, did not block the inhibition of LPL by eitherhANGPTL3(17-169)-mFc at 80 nM or mANGPTL3(17-240)-His at 500 nM:H1M896N+H4H1279P; H4H1250P+H4H1279P; H4H1248P+H4H1292P; andH4H1263S+H4H1292P. In this same assay, H4H1276S alone blocked these sameconstant concentrations of human and mouse ANGPTL3 with IC50s of 33 nMand 64 nM, respectively.

Example 7.1 In Vivo Effect of Anti-ANGPTL3 Antibody on Serum LipidLevels

The effect of the anti-hANGPTL3 antibody H4H1726S on serum lipid levelswas determined in C57BI/6 mice. Mice were pre-bled 7 days before theexperiment and put into groups of six mice each for each antibody dosetested. Antibodies were administered at 5 mg/kg (H4H1726S) and 10 mg/kg[H4H1726S and isotype-matched hIgG4(S108P) control with irrelevantspecificity] dose levels by subcutaneous injection on day 0 of thestudy. Mice were bled after 4 hours of fasting at days 1, 4, 7 and 12after antibody injections and serum lipid levels (triglycerides, totalcholesterol, non-HDL cholesterol, LDL cholesterol and HDL cholesterol)were determined in the serum by an ADVIA® 1800 Chemistry System(Siemens). Averages were calculated for each of the time points for eachantibody. Results, expressed as (mean±SEM) of serum lipid concentration,are shown in Tables 10-14.

TABLE 10 Serum triglycerides (mg/dL) Control Ab H4H1276S H4H1276S Daysafter (10 mg/kg) (5 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM MeanSEM −7 87.83 6.18 89.83 3.65 87.17 5.062 1 123.16 7.02 68.00 2.84 53.832.52 4 99.66 10.15 62.16 5.82 50.67 3.51 7 99.83 4.57 55.83 4.95 39.672.55 12 82.00 5.75 76.83 10.56 53.00 6.51

TABLE 11 Total cholesterol (mg/dL) Control Ab H4H1276S H4H1276S Daysafter (10 mg/kg) (5 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM MeanSEM −7 82.50 2.11 80.33 1.15 81.33 2.14 1 87.83 1.87 71.50 5.48 63.673.38 4 75.00 2.58 59.50 3.51 51.00 2.98 7 83.50 1.77 67.00 1.79 61.332.33 12 87.83 1.82 83.00 4.30 69.33 3.22

TABLE 12 Non-HDL cholesterol (mg/dL) Control Ab H4H1276S H4H1276S Daysafter (10 mg/kg) (5 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM MeanSEM −7 41.18 0.75 38.78 0.81 40.23 1.18 1 42.18 0.55 35.75 3.05 32.701.94 4 36.40 1.04 29.63 2.16 27.55 1.78 7 40.82 0.75 34.67 1.83 32.021.68 12 41.72 0.87 39.85 2.21 35.13 1.47

TABLE 13 LDL cholesterol (mg/dL) Control Ab H4H1276S H4H1276S Days after(10 mg/kg) (5 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM Mean SEM −74.68 0.35 4.40 0.34 4.47 0.21 1 5.40 0.41 5.20 0.79 5.33 0.71 4 4.800.45 4.88 0.67 5.33 0.73 7 5.38 0.46 5.83 0.48 6.40 0.67 12 5.67 0.596.12 0.65 5.35 0.48

TABLE 14 HDL cholesterol (mg/dL) Control Ab H4H1276S H4H1276S Days after(10 mg/kg) (5 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM Mean SEM −741.32 1.57 41.55 0.90 41.10 1.37 1 45.65 1.85 35.75 2.54 30.97 2.13 438.60 2.26 29.87 1.62 23.45 1.66 7 42.68 1.81 32.33 1.25 29.32 1.72 1246.12 1.94 43.15 2.52 34.20 1.99

Levels of circulating H4H1726S (Serum Ab) were also determined using astandard ELISA assay. Briefly, plates were coated with a goat anti-humanFc antibody (Sigma-Aldrich) to capture Serum Ab. Serum was then added tothe plates and captured human antibody was detected by chemiluminescenceusing a horseradish peroxidase (HRP) conjugated goat anti-human IgGantibody (Sigma-Aldrich). Results, expressed as (mean±SEM) of are shownin Table 15. Control: Mice that received an isotype-matched Control Ab.

TABLE 15 Serum Ab (μg/mL) Control Ab H4H1276S H4H1276S Days after (10mg/kg) (5 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM Mean SEM 1 65.008.05 36.38 7.57 126.23 9.96 4 59.16 4.94 29.91 4.32 86.28 6.77 7 58.236.02 30.86 5.11 54.24 8.96 12 41.35 9.76 5.48 1.79 39.04 7.08

Single administration of H4H1276S to C57BI/6 mice at 10 mg/kg led to˜60% reduction in circulating triglycerides 7 days after the antibodyadministration (compared to isotype control). The administration ofH4H1276S also led to a significant reduction in total cholesterol,non-HDL cholesterol and HDL cholesterol and had no effect on LDLcholesterol. A reduction in lipid levels was also observed, but lesspronounced, at the 5 mg/kg compared to 10 mg/kg dose levels; e.g., serumtriglycerides were reduced by 44% (compared to isotype control) 7 daysafter antibody administration.

Example 7.2 In Vivo Effect of Anti-ANGPTL3 Antibodies on Serum LipidLevels

The evaluation of the in vivo effects of anti-hANGPTL3 antibodiesH4H1276S and comparator antibody 4.9.1 on serum lipid levels wasconducted in C57BI/6 mice. Antibody 4.9.1 was prepared based on theamino acid sequences of SEQ ID No: 24 (VH) and SEQ ID No: 32 (VL) asdisclosed in US Patent Application Publication No. 2008/0177045 and as amouse IgG1 isotype. Mice were pre-bled 7 days before the experiment andput into groups of six mice per group. Antibodies H4H1276S, 4.9.1, andisotype-matched negative controls (human IgG4 and mouse IgG1,respectively) with irrelevant specificity were administered at 10 mg/kgdose by subcutaneous injection on day 0 of the study. Mice were bledafter 4 hours of fasting at days 1, 7, 11 and 20 after injection ofantibodies, and serum lipids levels (triglycerides, total cholesterol,non-HDL cholesterol, LDL cholesterol and HDL cholesterol) weredetermined in the serum using an ADVIA® 1800 Chemistry System (Siemens).Average lipid concentrations were calculated for each of the time pointfor each antibody. Results, expressed as (mean±SEM) of serum lipidconcentration, are shown in Tables 16-20.

TABLE 16 Serum triglycerides (mg/dL) Days after Control (IgG4) H4H1276SControl (IgG1) 4.9.1 injection Mean SEM Mean SEM Mean SEM Mean SEM −7109.16 9.05 109.16 6.44 112.80 6.87 109.17 7.24 1 81.67 6.76 46.00 3.5995.20 8.92 41.83 2.42 7 95.67 5.42 49.67 3.86 101.80 7.55 96.00 3.70 11100.83 6.20 51.00 5.89 117.00 6.00 92.00 4.50 20 82.17 4.36 72.67 3.4779.40 6.59 73.83 5.03

TABLE 17 Total cholesterol (mg/dL) Days after Control (IgG4) H4H1276SControl (IgG1) 4.9.1 injection Mean SEM Mean SEM Mean SEM Mean SEM −780.81 0.95 80.92 3.05 80.32 2.84 79.37 2.76 1 82.82 2.11 67.33 3.6082.98 2.17 71.35 1.82 7 79.20 1.81 63.58 3.98 85.02 7.27 82.07 4.36 1189.97 3.18 69.02 2.11 83.92 2.49 84.58 1.08 20 92.43 1.10 80.17 3.2087.47 2.58 88.40 2.84

TABLE 18 Non-HDL cholesterol (mg/dL) Days after Control (IgG4) H4H1276SControl (IgG1) 4.9.1 injection Mean SEM Mean SEM Mean SEM Mean SEM −742.44 1.18 42.87 1.03 43.20 2.44 41.73 1.40 1 40.85 1.48 35.33 1.7940.68 0.87 36.97 1.49 7 39.03 1.04 33.72 2.86 43.30 4.35 40.47 2.35 1144.68 1.93 35.18 1.64 40.28 0.95 41.38 1.05 20 47.40 0.67 42.10 1.5144.72 1.66 44.40 1.57

TABLE 19 LDL cholesterol (mg/dL) Days after Control (IgG4) H4H1276SControl (IgG1) 4.9.1 injection Mean SEM Mean SEM Mean SEM Mean SEM −73.93 0.07 4.20 0.26 4.38 0.26 4.20 0.18 1 3.95 0.28 4.25 0.37 3.92 0.174.62 0.37 7 3.75 0.14 5.25 1.08 5.76 1.61 4.57 0.73 11 5.05 0.26 5.470.23 4.88 0.27 4.78 0.23 20 5.72 0.34 4.95 0.32 4.97 0.28 5.65 0.46

TABLE 20 HDL cholesterol (mg/dL) Days after Control (IgG4) H4H1276SControl (IgG1) 4.9.1 injection Mean SEM Mean SEM Mean SEM Mean SEM −738.37 0.95 38.00 2.27 37.12 1.88 37.63 1.52 1 41.97 1.32 32.00 1.8942.30 2.09 34.38 0.85 7 40.17 0.93 29.87 1.23 41.72 2.97 41.60 2.47 1145.28 1.80 33.83 1.15 43.64 1.70 43.20 1.57 20 45.03 0.75 38.07 1.7942.75 1.69 44.00 1.83

A single 10 mg/kg dose of H4H1276S in C57BI/6 mice resulted in reductionof plasma triglyceride levels compared to isotype control on days 1, 7,11 and 20 after antibody injection; and this effect was more sustainedcompared to a single treatment at the same dose level with thecomparator 4.9.1 (Table 16). Administration of H4H1276S also led to areduction in total cholesterol (Table 17) and HDL cholesterol (Table 20)in C57BI/6 mice.

Example 8 In Vivo Effect of H4H1276S on Serum Lipid Levels inHyperlipidemic ApoE^(−/−) Mice

The effect of anti-hANGPTL3 antibody H4H1276S on serum lipids levels wasdetermined in apoE^(−/−) mice. These mice are hyperlipidemic with themajority of their circulating cholesterol found in the form of VLDL andLDL. Mice were pre-bled 7 days before the experiment and put into groupsof six mice per group. The antibodies, H4H1276S and an isotype-matched(hIgG4) control with irrelevant specificity, were administered at 10mg/kg dose by subcutaneous injection on day 0 of the study. Mice werebled after 4 hours of fasting at days 1, 4, 7 and 11 after injection ofantibodies; and serum lipids levels (triglycerides, total cholesterol,non-HDL cholesterol, LDL cholesterol and HDL cholesterol) weredetermined in the serum using an ADVIA® 1800 Chemistry System (Siemens).Average lipid concentrations were calculated for each of the time pointsfor each antibody-treated group. Results, expressed as (mean±SEM) ofserum lipid concentration, are shown in Tables 21-25.

TABLE 21 Serum triglycerides (mg/dL) Days after Control (hIgG4) H4H1276Sinjection Mean SEM Mean SEM −7 134.17 11.81 141.67 17.14 1 156.33 19.0661.33 3.66 4 181.00 7.70 70.50 4.46 7 190.67 27.65 52.50 6.22 11 170.0028.85 133.00 13.56

TABLE 22 Total cholesterol (mg/dL) Days after Control (hIgG4) H4H1276Sinjection Mean SEM Mean SEM −7 450.67 25.68 479.33 13.76 1 497.50 37.77386.33 28.59 4 395.00 14.37 281.20 20.83 7 447.33 22.18 295.50 12.86 11463.80 36.01 398.03 23.13

TABLE 23 Non-HDL cholesterol (mg/dL) Days after Control (hIgG4) H4H1276Sinjection Mean SEM Mean SEM −7 435.87 25.59 464.53 13.97 1 476.30 37.29371.25 28.65 4 375.61 14.51 266.26 21.19 7 427.66 21.45 280.75 12.55 11442.27 34.19 379.55 22.31

TABLE 24 LDL cholesterol (mg/dL) Days after Control (hIgG4) H4H1276Sinjection Mean SEM Mean SEM −7 14.27 1.63 14.87 0.90 1 17.42 2.94 11.231.81 4 10.28 1.52 6.62 0.83 7 11.82 1.40 6.32 0.45 11 13.90 2.54 10.211.14

TABLE 25 HDL cholesterol (mg/dL) Days after Control (hIgG4) H4H1276Sinjection Mean SEM Mean SEM −7 14.80 0.37 14.80 0.54 1 21.20 1.00 15.080.53 4 19.33 0.94 14.53 0.75 7 19.77 0.78 14.58 0.72 11 21.53 1.89 18.481.00

Single administration of H4H1276S to apoE^(−/−) mice at 10 mg/kg led to˜72% (mean) reduction in circulating triglycerides (Table 21) and ˜46%(mean) reduction in LDL cholesterol (Table 24) 7 days after the antibodyadministration (compared to the isotype-matched control Ab, i.e.,hIgG4). The administration of H4H1276S also led to a reduction in totalcholesterol (Table 22) and non-HDL cholesterol (Table 23).

Levels of circulating H4H1276S (Serum Ab) were also determined using astandard ELISA assay. Briefly, plates were coated with a goat anti-humanFc antibody (Sigma-Aldrich) to capture Serum Ab. Serum was then added tothe plates and captured antibodies were detected by chemiluminescenceusing a horseradish peroxidase (HRP) conjugated goat anti-human IgGantibody (Sigma-Aldrich). Results, expressed as (mean±SEM), are shown inTable 26 (Control: mice that received an isotype-matched control Ab,hIgG4).

TABLE 26 Serum Ab (μg/mL) Control (hIgG4) H4H1276S Days after (10 mg/kg)(10 mg/kg) injection Mean SEM Mean SEM 1 89.98 16.70 115.29 19.75 467.18 2.38 86.61 5.32 7 58.52 2.00 39.85 6.91 12 43.26 1.76 3.18 2.64

Example 9 In Vivo Effect of H4H1276S on Circulating Lipid Levels inHyperlipidemic Ldlr^(−/−) Mice

The effect of the anti-hANGPTL3 antibody H4H1726S on serum lipid levelswas determined in Ldlr^(−/−) mice. These mice are hyperlipemic with amajority of circulating cholesterol found in the form of LDL due to thelack of LDLR, the major receptor for LDL cholesterol uptake.

Mice were pre-bled 7 days before the experiment and put into groups ofsix mice. The antibodies, H4H1726S and isotype-matched (hIgG4) negativecontrol, were administered at 10 mg/kg dose by subcutaneous injection onday 0 of the study. Mice were bled after 4 hours of fasting at days 1,4, 7 and 11 after antibody injection and serum lipids levels(triglycerides, total cholesterol, non-HDL cholesterol, LDL cholesteroland HDL cholesterol) were determined using an ADVIA® 1800 ChemistrySystem (Siemens) clinical chemistry analyzer. Averages were calculatedfor each time point for each antibody. Results, expressed as (mean±SEM)of serum lipids concentration (triglycerides, total cholesterol, non-HDLcholesterol, LDL cholesterol and HDL cholesterol), are shown in Tables27-31, respectively. (Control=mice that received an isotype-matchedcontrol antibody).

TABLE 27 Serum triglycerides (mg/dL) Antibody Control H4H1726S Daysafter (10 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM −7 114.50 11.08110.83 6.89 1 131.50 6.18 74.17 3.30 4 112.67 8.94 68.00 3.91 7 136.6711.55 92.67 12.16 11 142.33 7.10 95.83 8.67

TABLE 28 Total cholesterol (mg/dL) Antibody Control H4H1726S Days after(10 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM −7 237.95 7.33 236.995.68 1 241.97 10.58 206.98 9.68 4 229.88 7.61 172.96 4.49 7 234.74 10.49176.28 7.47 11 251.87 18.82 201.73 10.12

TABLE 29 Non-HDL cholesterol (mg/dL) Antibody Control H4H1726S Daysafter (10 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM −7 180.81 7.47182.79 5.93 1 184.35 10.22 155.93 8.74 4 175.13 7.26 130.79 4.66 7174.84 9.26 126.56 6.63 11 190.00 17.07 145.43 7.34

TABLE 30 LDL cholesterol (mg/dL) Antibody Control H4H1726S Days after(10 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM −7 62.75 2.18 62.751.81 1 63.25 2.40 53.82 4.09 4 60.97 3.14 49.65 2.72 7 59.52 2.99 46.052.13 11 63.23 3.07 54.28 1.67

TABLE 31 HDL cholesterol (mg/dL) Antibody Control H4H1726S Days after(10 mg/kg) (10 mg/kg) injection Mean SEM Mean SEM −7 57.13 1.56 54.201.89 1 57.62 0.88 51.05 0.98 4 54.75 2.23 42.17 1.89 7 59.90 2.51 49.722.35 11 61.87 2.48 56.30 3.43

As shown in Tables 27-31, administration of H4H1726S to Ldlr^(−/−) miceled to a significant reduction in plasma triglycerides with a maximalobserved reduction of 44% (based on mean values). Significant reductionsin LDL cholesterol (up to 23%), as well as total cholesterol, non-HDLcholesterol and HDL cholesterol, were also observed in H4H1726S-treatedsubjects. Reduction of LDL cholesterol in mice deficient for the majorreceptor for LDL cholesterol uptake (LDLR) suggests an LDLR-independentmechanism for LDL cholesterol reduction by ANGPTL3 inhibition.

Levels of circulating H4H1726S (Serum Ab) were also determined using astandard ELISA assay. Briefly, plates were coated with a goat anti-humanFc antibody (Sigma-Aldrich) to capture Serum Ab. Serum was then added tothe plates and captured antibodies were detected by chemiluminescenceusing a horseradish peroxidase (HRP) conjugated goat anti-human IgGantibody (Sigma-Aldrich). Results, expressed as (mean±SEM) of are shownin Table 32. (Control=mice that received an isotype-matched controlantibody).

TABLE 32 Serum Ab (μg/mL) Antibody Control H4H1726S Days after (10mg/kg) (10 mg/kg) injection Mean SEM Mean SEM 1 44.59 1.95 58.79 5.95 442.28 6.12 47.21 10.24 7 41.76 3.87 28.88 5.97 11 37.25 6.85 21.02 4.86

As shown in Table 32, serum levels of H4H1726S decreased to about 21μg/mL by day 11 following injection of mice with 10 mg/kg of antibody.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

We claim:
 1. An isolated human antibody or antigen-binding fragmentthereof that specifically binds human angiopoietin-like protein 3(hANGPTL3) of SEQ ID NO:161 and neutralizes, reduces, or interfereswith, at least one activity of hANGPTL3.
 2. The antibody or fragment ofclaim 1, wherein the antibody or fragment thereof binds to an epitopesituated within the N-terminal coiled-coil region at residues 17 to 209of SEQ ID NO:161.
 3. The antibody or fragment of claim 2, wherein theantibody or fragment thereof does not bind to the ANGPTL3 peptide of SEQID NO:170.
 4. The antibody or fragment of any one of claim 2, whereinthe epitope is situated within residues 17 to 200, 17 to 100, 17 to 70,17 to 65, 17 to 60, 17 to 57, 17 to 50, 40 to 200, 40 to 100, 40 to 70,50 to 200, 50 to 100, 50 to 70, 58 to 200, 58 to 100, 58 to 70, 58 to68, or 61 to 66, of SEQ ID NO:161.
 5. The antibody or fragment of anyone of claim 1, wherein the antibody or fragment is a chimeric or fullyhuman antibody or antibody fragment.
 6. The antibody or fragment of anyone of claim 1, comprising a heavy chain variable region (HCVR) selectedfrom the group consisting of SEQ ID NO:2, 18, 34, 50, 66, 82, 98, 114,130, 146 and 180; and/or a light chain variable region (LCVR) selectedfrom the group consisting of SEQ ID NO:10, 26, 42, 58, 74, 90, 106, 122,138, 154 and
 188. 7. The antibody or fragment of claim 6, wherein theantibody or fragment thereof comprises a HCVR and LCVR sequence pair(HCVR/LCVR) selected from the group consisting of SEQ ID NO:2/10, 18/26,34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154 and180/188.
 8. The antibody or fragment of any one of claims 1, wherein theantibody or fragment comprises heavy chain complementarity determiningregion (CDR) sequences, HCDR1, HCDR2 and HCDR3, and light chain CDRsequences, LCDR1, LCDR2 and LCDR3, contained within a heavy chainvariable region (HCVR) and light chain variable region (LCVR) sequencepair (HCVR/LCVR) selected from the group consisting of SEQ ID NO:2/10,18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154 and180/188.
 9. The antibody or fragment of claim 8, wherein theHCDR1/HCDR2/HCDR3 sequence combination is selected from the groupconsisting of SEQ ID NO:4/6/8, 20/22/24, 36/38/40, 52/54/56, 68/70/72,84/86/88, 100/102/104, 116/118/120, 132/134/136, 148/150/152 and182/184/186; and/or the LCDR1/LCDR2/LCDR3 sequence combination selectedfrom the group consisting of SEQ ID NO:12/14/16, 28/30/32, 44/46/48,60/62/64, 76/78/80, 92/94/96, 108/110/112, 124/126/128, 140/142/144,156/158/160 and 190/192/194.
 10. The antibody or fragment of claim 9,wherein the antibody or fragment comprises aHCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequence combination selected fromthe group consisting of 4/6/8/12/14/16, 20/22/24/28/30/32,36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80,84/86/88/92/94/96, 100/102/104/108/110/112, 116/118/120/124/126/128,132/134/136/140/142/144, 148/150/152/156/158/160 and182/184/186/190/192/194.
 11. An antibody or antigen-binding fragmentthereof which competes for binding to hANGPTL3 with, or which binds thesame epitope on hANGPTL3 as, an antibody or antigen-binding fragmentcomprising a heavy chain and light chain complementarity determiningregion (CDR) sequence combination selected from the group consisting of4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64,68/70/72/76/78/80, 84/86/88/92/94/96, 100/102/104/108/110/112,116/118/120/124/126/128, 132/134/136/140/142/144,148/150/152/156/158/160 and 182/184/186/190/192/194.
 12. The antibody orantigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment cross-reacts with cynomolgus monkey ANGPTL3.13. The antibody or antigen-binding fragment of claim 1, wherein theantibody or antigen-binding fragment cross-reacts with mouse or ratANGPTL3.
 14. The antibody or antigen-binding fragment of claim 1,wherein the antibody or fragment cross-reacts with any of cynomolgusmonkey ANGPTL3, mouse ANGPTL3, and rat ANGPTL3.
 15. The antibody orantigen-binding fragment of claim 1, wherein the antibody fragment is asingle chain antibody, an Fab, or an F(ab′)₂.
 16. An isolated nucleicacid molecule encoding the antibody or antigen-binding fragment ofclaim
 1. 17. An expression vector comprising the nucleic acid moleculeof claim
 16. 18. An isolated host cell comprising the expression vectorof claim
 17. 19. A method of producing an anti-hANGPTL3 antibody orantigen-binding fragment thereof, comprising growing the host cell ofclaim 18 under conditions permitting production of the antibody orfragment thereof, and recovering the antibody or fragment thereof soproduced.
 20. A pharmaceutical composition comprising the antibody orantigen-binding fragment of claim 1, and a pharmaceutically acceptablecarrier.
 21. The pharmaceutical composition of claim 20, wherein theantibody or fragment thereof comprises a heavy chain and light chaincomplementarity determining region (CDR) sequence combination of SEQ IDNO:68/70/72/76/78/80, or a heavy chain variable region and a light chainvariable region HCVR/LCVR pair comprising SEQ ID NO:66/74.
 22. A methodfor preventing or treating a disease or disorder which is prevented,ameliorated, improved or inhibited by reduction or inhibition of ANGPTL3activity, the method comprising administering to a subject in needthereof a therapeutically effective amount of the pharmaceuticalcomposition of claim
 20. 23. The method of claim 22, further comprisingadministering to the subject one or more additional therapeutic agentsselected from the group consisting of an inhibitor of HMG-CoA reductase;an inhibitor of cholesterol uptake or bile acid re-absorption, or both;an agent which increases lipoprotein catabolism; an agent which reducesthe number of non-fatal heart attacks; and an activator of the LXRtranscription factor.
 24. The method of claim 22, further comprisingadministering to the subject one or more additional therapeutic agentsselected from the group consisting of a statin, niacin, fibrate,anti-hANGPTL4 antibody and anti-PCSK9 antibody.
 25. The method of claim22, wherein the disease or disorder is selected from the groupconsisting of hypertriglyceridemia, hypercholesterolemia,chylomicronemia, atherogenic dyslipidemia, a cardiovascular disease ordisorder, acute pancreatitis, nonalcoholic steatohepatitis (NASH),diabetes and obesity.
 26. A method of enhancing the clearance ofhANGPTL3 from the circulation of a subject in need thereof, the methodcomprising administering to the subject at least two antibodies orantigen-binding fragments thereof that do not compete with one anotherfor binding to hANGPTL3 and do not block at least one activity ofhANGPTL3.
 27. The method of claim 26, wherein the at least twoantibodies or antigen-binding fragments do not block lipoprotein lipase(LPL) activity of ANGPTL3.
 28. The method of claim 27, wherein the twoantibodies or antigen-binding fragments thereof comprise HCVR/LCVR aminoacid sequence pairs selected from the group consisting of: (i) SEQ IDNO:82/90 and 180/188; (ii) SEQ ID NO:82/90 and 18/26; (iii) SEQ IDNO:114/122 and 180/188; and (iv) SEQ ID NO:114/122 and 18/26.